Method and compounds for promoting healing and reducing inflammation

ABSTRACT

This invention relates to methods of promoting healing and reducing inflammation, and compositions therefore. In particular, the invention relates to the use of 1,3-dialkyl-4,5-bis(N-methylcarbamoyl)imidazolium salts to promote wound healing and to reduce inflammation. Novel compounds and compositions are also provided. In one preferred embodiment the invention provides a method of treatment of myocardial infarction.

This invention relates to methods of promoting healing and reducinginflammation, and compositions therefore. In particular, the inventionrelates to the use of 1,3-dialkyl-4,5-bis(optionally N-substitutedcarbamoyl)imidazolium salts to promote healing and to reduceinflammation. In one preferred embodiment the compounds are useful inthe treatment of myocardial infarction. Novel compounds and compositionsare also provided.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

Tissue or organ damage in mammals is usually accompanied byinflammation. Inflammation is a complex process associated with theactivity of inflammatory mediators such as histamine, serotonin,bradykinin, prostaglandin and other biologically active substances, andmay be caused by a variety of endogenous or exogenous pathologicalagents or processes. The inflammatory process may result in themalfunction of specific organs and in deterioration of the overall stateof health.

Both non-steroidal anti-inflammatory agents (NSAIDS) and steroidhormones such as corticosteroids have an anti-inflammatory effect, andare widely used in medical practice. Corticosteroids have seriousside-effects, including excessive accumulation of sodium and water inthe body, oedema, increased blood pressure, decreased resistance toinfection, ulceration of the gastrointestinal mucosa, impaired woundhealing and tissue regeneration, susceptibility to blood clotting, andobesity, as well as endocrine, nervous and psychiatric disorders. Ifcorticosteroids are administered over a long period, there is a riskthat the production of natural hormones by the adrenal glands can besuppressed.

The anti-inflammatory activity of NSAIDS is due to their suppressiveeffect on the enzymes involved in the synthesis of prostaglandin(prostaglandin synthetase), serotonin (5-hydroxytryptophandecarboxylase), histamine (histamine decarboxylase) and on the releaseof inflammatory mediators. NSAIDS may cause side-effects such asirritation of the gastric mucosa, allergic reactions, and liver andkidney dysfunction.

Wound healing is a complex morphological, pathophysiological andbiochemical process whose progress and outcome are significantlyaffected by a variety of factors relating to skin injury. The generalwound healing process involves three stages:

1. Degradation of necrotic pulp and removal of necrotic material fromthe wound defect via an inflammatory process;

2. Proliferation of connective tissue elements and formation ofgranulation tissue to fill the wound defect; and

3. Fibrosis of the granulation tissue, and formation andepithelialization of scar tissue.

Many agents have been proposed for stimulation of wound healing and skinregeneration, including hormones, enzymes, plant-derived agents, andpolypeptide growth factors.

Wounds can result not only from trauma, including burns, but also fromsurgical interventions. In particular, plastic reconstructive surgeryand dermatological techniques can result in skin defects which requirerapid healing with no or minimal scar formation. These techniquesinclude procedures such as laser skin resurfacing, blepharoplasty,dermabrasion, chemical peeling and the like. During the post-operativeperiod, patients may suffer complications such as prolonged erythema,dermatitis, hyperpigmentation, or infection with organisms such asPseudomonas aeruginosa, Staphyloccoccus aureus, S. epidermidis, someGram-negative bacteria, various types of Candida, or the virus Herpessimplex. This may result in formation of hypertrophic scars, and maynecessitate a long treatment period (Sriprachya-Anunt et al, 1997).

While these complications may be reduced or avoided by carefulpost-operative care and the use of anti-infective agents and promotersor tissue repair and regeneration, complete avoidance of complicationsis not always possible.

To heal skin wounds, various drugs may be used as stimulators of thetissue regeneration process, including proteolytic enzymes, syntheticanabolic hormones, growth factors, antiseptics, honey and its products,phytomedicinals, various oils, adsorbents, etc (Mashkovsky, 1998).

In the post-operative period antibiotics, antiviral drugs,corticosteroids, and vitamins such as vitamins A, C, and E may be usedin conjunction with drugs enhancing tissue repair and regeneration(Katzung, 1998).

Thus there is still a considerable need in the art for methods andagents for effective and low cost stimulation of wound healing andreduction of scar formation.

The compound ethimizole (1-ethylimidazole-4,5-dicarboxylic acidbis-N-methylamide; 1-ethyl-4,5-di(N-methylcarbamoyl)imidazole)

has been used as an antiallergic and anti-inflammatory medicinal agentin the treatment of arthritis, inflammatory polyarthritis, and certainforms of bronchial asthma (M. D. Mashkovsky, Medicinals, Medicine, 1987,pp. 130-131). Under biological conditions, the uncharged imidazole ringof imidazole-4,5-dicarboxylic acid enables ethimizole to penetratethrough the blood-brain barrier.

Consequently this drug causes undesirable side effects associated withits influence on the central nervous system. In particular, ethimizoleis contraindicated for patients with motor or psychological agitation.Ethimizole has not been suggested to be useful for treatment of wounds.

In our previous patent RU1075668, we disclosed1,3-dialkyl-4,5-bis(N-methylcarbamoyl)imidazole benzene sulphonatecompounds which had a stimulating effect on tissue energy metabolism.These compounds had the general formula

in which R¹ and R² may be the same or different, and are independentlyselected from methyl and ethyl, and X⁻ is benzenesulphonate. In thesecompounds the imidazole ring of ethimizole is replaced by a chargedimidazolium ring. Such charged compounds are unable to penetrate throughthe blood-brain barrier, and therefore cannot affect the central nervoussystem.

The specification of RU1075668 disclosed the synthesis of threecompounds within the formula, namely the 1,3-dimethyl,1-methyl-3-ethyl,and 1,3-diethyl compounds, and their ability to prevent development ofneurogenic gastric lesions in rats, to promote healing of such lesions,and to increase creatine phosphate levels in the gastric wall, whenadministered intra-peritoneally. It was suggested that, because of thisstimulatory effect on energy metabolism, the compounds might be usefulas tissue repair agents.

However, no guidance at all was provided as to how any other conditioncould be treated, how the compounds should be formulated, or by whatroutes they should be administered. Only intra-peritoneallyadministration was disclosed. In particular, there was no generaldisclosure or suggestion that any of the compounds disclosed in thisspecification could have any activity in promoting healing of wounds,burns, skin ulcers or the like, in reducing scar formation, in reducinginflammation, in stimulating repair of bone, or in treating myocardialinfarction.

We have now found that 1,3-dialkyl-4,5-bis(N-methylcarbamoyl)imidazoliumsalts promote tissue repair in a variety of settings, and in particularpromote wound healing and reduce scar formation. We have also found thatthat a number of 1,3-dialkyl-4,5-bis(N-methylcarbamoyl)imidazolium saltspossess anti-inflammatory and wound healing properties, and that thecompounds are active both orally and topically. These compoundsdemonstrate an anti-inflammatory effect in experimental models ofinflammation, have no toxic effects in a variety of assays, and arereadily synthesised using simple reaction schemes.

Without wishing to be limited by any proposed mechanism, we believe thattheir anti-inflammatory activity is due to their suppressive effect onthe synthesis and secretion of mediators of inflammation.

SUMMARY OF THE INVENTION

In a first general aspect, the invention provides a method of promotingtissue repair or wound healing, comprising the step of administering aneffective amount of a 1,3-dialkyl-4,5-bis(optionally N-substitutedcarbamoyl)imidazolium salt to a subject in need of such treatment.

In one preferred embodiment, the invention provides a method of reducinginflammation, comprising the step of administering an effective amountof a 1,3-dialkyl-4,5-bis (optionally N-substituted carbamoyl)imidazoliumsalt to a subject in need of such treatment.

In a second preferred embodiment, the invention provides a method ofreducing scar formation, comprising the step of administering aneffective amount of a 1,3-dialkyl-4,5-bis (optionally N-substitutedcarbamoyl)imidazolium salt to a subject in need of such treatment.

In a third preferred embodiment, the invention provides a method oftreatment of myocardial infarction, comprising the step of administeringan effective amount of a 1,3-dialkyl-4,5-bis (optionally N-substitutedcarbamoyl)imidazolium salt to a subject in need of such treatment.

In a fourth preferred embodiment, the invention provides a method ofstimulating bone repair, comprising the step of administering aneffective amount of a 1,3-dialkyl-4,5-bis (optionally N-substitutedcarbamoyl)imidazolium salt to a subject in need of such treatment.

In a fifth preferred embodiment, the invention provides a method oftreatment of ulcerative colitis, comprising the step of administering aneffective amount of a 1,3-dialkyl-4,5-bis (optionally N-substitutedcarbamoyl)imidazolium salt to a subject in need of such treatment.

Preferably the 1,3-dialkyl-4,5-bis (optionally N-substitutedcarbamoyl)imidazolium salt is a compound of formula I

in which R¹ and R² may be the same or different, and each is selectedfrom the group consisting of hydrogen and a linear or branched alkylgroup of 1 to 6 carbon atoms, which may optionally be substituted by anamino, substituted or unsubstituted aminomethyl, nitro, hydroxyl,halogen, carboxy, or carboxylic acid amide group;

R³ and R⁴ may be the same or different, and each is a linear or branchedalkyl group of 1 to 6 carbon atoms; and

X⁻ is a pharmaceutically acceptable inorganic or organic anion selectedfrom the group consisting of chloride, bromide, iodide, sulphate,nitrate, phosphate, perchlorate, formate, acetate, fumarate, malate,malonate, citrate, benzoate, salicylate, benzenesulfonate,methylsulfonate, p-toluenesulfonate, gentisate (dihydroxybenzoate), andnaphthalene-8-sulfonate.

When R¹ or R² is a halogen, this halogen is chlorine, bromine or iodine.

Preferably R¹ and R² are different, for example if R¹ is hydrogen, R² isR² is an alkyl group with 1 to 6 carbon atoms; R³ and R⁴ are the same ordifferent, and are each independently an alkyl group with 1 to 6 carbonatoms, more preferably 1 to 4 carbon atoms. Even more preferably R³ ismethyl and R⁴ is ethyl.

The anion X⁻ has no specific limitations, and is preferably an organicanion selected from the group consisting of benzenesulfonate,methylsulfonate, n-toluenesulfonate, formate, acetate, fumarate, malate,malonate, citrate, benzoate, salicylate, gentisate, andnaphthalene-8-sulfonate. More preferably X⁻ is benzenesulfonate,benzoate, salicylate, or gentisate. Most preferably X⁻ isbenzenesulphonate. Alternatively X⁻ is preferably an inorganic anionselected from the group consisting of chloride, bromide, and iodide.

R³ and/or R⁴ may optionally be substituted with a group selected from asubstituted or unsubstituted amino, substituted or unsubstitutedaminomethyl, NO₂, acetamide, or substituted or unsubstitutedsulphonamide group. Preferably at least one of R³ and R⁴ isunsubstituted; more preferably both R³ and R⁴ are unsubstituted. WhereR³ or R⁴ is a substituted sulphonamide, the substituent is preferably analkyl chain of 1 to 6, more preferably 1 to 4 carbon atoms.

Particularly preferred compounds for use in the method of the inventionare:

-   1,3-dimethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzenesulfonate    (1),-   1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium    benzenesulfonate (2),-   1,3-diethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzenesulfonate    (3),-   1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium chloride (4)-   1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzoate (5),-   1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium salicylate    (6), and-   1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium gentisate    (7).

The method of the invention is applicable to the treatment of damage toepithelial, mucosal, muscular, cardiac, liver, and bone tissue, causedby erosions, ulcers, chronic injury, infection, trauma or surgery. Forexample, the damage may be gastric or duodenal ulcers, myocardialinfarction, liver conditions such as cirrhosis and hepatitis, or bonefractures.

Wounds arising from a wide variety of causes can be treated using themethod of the invention, including but not limited to traumatic wounds,surgical wounds, burns, dehisced surgical incisions, grafts, diabeticulcers, varicose ulcers, decubitus ulcers (bedsores), trophic ulcers,tropical ulcers, steroid ulcers, indolent ulcers, oral or pharyngealulcers, aphthous ulcers, and corneal ulcers; gastric, duodenal, andpeptic ulcers; ulcerative colitis; cervical erosions; myocardial damage,including myocardial infarction; liver damage, for example caused bycirrhosis or hepatitis; and bone fractures.

In all embodiments of the first aspect of the invention, the1,3-dialkyl-4,5-bis(optionally N-substituted carbamoyl)imidazolium saltis preferably administered orally or, if appropriate for the specificcondition, topically or via enema.

In a second aspect, the invention provides a compound of formula I asdefined above, but with the proviso that when X⁻ is benzenesulphonate,R¹ is hydrogen and R² is methyl, then R³ and R⁴ are not methyl or ethyl.

In a third aspect, the invention provides a composition comprising acompound according to the invention, together with a pharmaceutically orveterinarily acceptable carrier. We have unexpectedly found that thecompounds of the invention are active when administered orally ortopically. Therefore in one preferred embodiment the composition isadapted for oral administration. In a second preferred embodiment thecomposition is one which is adapted to topical administration, such asan ointment, cream or gel.

Methods and pharmaceutical carriers for preparation of pharmaceuticalcompositions are well known in the art, as set out in textbooks such asRemington's Pharmaceutical Sciences, 19th Edition, Mack PublishingCompany, Easton, Pa., USA.

Examples of formulations which can be used for the purposes of theinvention include topical formulations such as creams, gels, ointments,and impregnated bandages; rectal or vaginal formulations such astampons, suppositories and pessaries; oral formulations such as tablets,lozenges, capsules or solutions; buccal or sublingual formulations suchas tablets or lozenges; solution or spray formulations for intranasaluse; enemas for rectal use; solution formulations for injection; andcellulose or collagen dressings.

It will be clearly understood that the proviso to formula I set out inthe second aspect of the invention applies only to simple solutionformulations in which the carrier is water or saline. It does not applyto the first aspect of the invention, or to compositions adapted fortopical or oral administration.

The mammal to be treated may be a human, or may be a domestic orcompanion animal. While it is particularly contemplated that thecompounds of the invention are suitable for use in medical treatment ofhumans, they are also applicable to veterinary treatment, includingtreatment of companion animals such as dogs and cats, and domesticanimals such as horses, cattle and sheep, or zoo animals such asnon-human primates, felids, canids, bovids, and ungulates.

The compounds and compositions of the invention may be administered byany suitable route, and the person skilled in the art will readily beable to determine the most suitable route and dose for the condition tobe treated. Dosage will be at the discretion of the attendant physicianor veterinarian, and will depend on the nature and state of thecondition to be treated, the age and general state of health of thesubject to be treated, the route of administration, and any previoustreatment which may have been administered.

The carrier or diluent, and other excipients, will depend on the routeof administration, and again the person skilled in the art will readilybe able to determine the most suitable formulation for each particularcase.

It will be clearly understood that the method of the invention may beused in conjunction with one or more other treatments, such as othertherapeutic agents or the use of hyperbaric oxygen or subatmosphericpressure.

In a fourth aspect the invention provides a method of synthesis of acompound of formula I, comprising the step of subjecting an1-alkyl-4,5-bis(optionally N-substituted carbamoyl)imidazole toalkylation (quaternization) with an alkyl benzenesulfonate to producethe corresponding imidazolium benzenesulfonate, and optionally replacingthe benzenesulfonate anion by ion exchange, in which the imidazolemoiety is as defined in formula I.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the healing of skin full-thickness wounds at 15 days.

-   -   A: control group, self-healing without treatment;    -   B: experimental group treated with compound (2) (10% cream);    -   C: control group treated with Spasatel balm;    -   D: experimental group treated with Solcoseryl gel.

FIG. 2 illustrates the healing of cryogenic damage to the mucousmembrane of the mouth.

-   -   A: controls, cryogenic damage—7 days;    -   B: experimental group treated with compound (2), 50 mg/kg—7        days;    -   C: experimental group treated with compound (2), 50 mg/kg—14        days.

FIG. 3 compares the effect of pharmacological agents on gastric ulcer inrats following electrostimulation. The vertical axis represents thenumber of ulcers per animal:

-   -   1. Control    -   2. Animals treated with 50 mg/kg methyl uracil.    -   3. Animals treated with 12 mg/kg cimetidine.    -   4. Animals treated with mg/kg Compound 2.    -   Panel A: Intraperitoneal administration; Panel B: oral        administration.

FIG. 4 illustrates the healing of cryogenic ulcers of the largeintestine, at 7 days.

-   -   A: controls, cryogenic ulcer;    -   B: experimental group treated with compound (2), 20 mg/kg;    -   C: experimental group treated with compound (2), 50 mg/kg;    -   D: experimental group treated with methyl uracil 180 mg/kg.

FIG. 5 shows the effect of Compound 2 on cyclic AMP levels (panel A) andCa⁺⁺ levels (panel B) in rabbit myocardial tissue.

-   -   1. Untreated control.    -   2. Electrostimulation alone.    -   3. 10 mg/kg Compound 2 prior to electrostimulation    -   4. Compound 2 given 3 days after electrostimulation.    -   5. Compound 2 daily for 3 days after electrostimulation.

FIG. 6A shows creatine kinase levels in rabbit myocardium (a) and bloodplasma (b) after electrostimulation of the aortic arch.

FIG. 6B shows the effect of Compound 2 on creatine phosphate levels inthe myocardium. In both FIGS. 6A and 6B:

-   -   1. Untreated control animals.    -   2. Electrostimulation alone.    -   3. Animals treated with 10 mg/kg Compound 2.    -   4. Animals treated with 10 mg/kg riboxine.

FIG. 7 illustrates regeneration of bone tissue in the area of anartificially-created defect in the bone plate of the lower jaw of rats.

-   -   A: controls, self-healing without treatment—1 month;    -   B: experimental group treated with compound (2) 50 mg/kg—3        months;    -   C: controls, self-healing without treatment—3 months;    -   D: experimental group treated with compound (2) 100 mg/kg—3        months.

DETAILED DESCRIPTION OF THE INVENTION

In the claims which follow and in the preceding description of theinvention, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprise” or variationssuch as “comprises” or “comprising” is used in an inclusive sense, i.e.to specify the presence of the stated features but not to preclude thepresence or addition of further features in various embodiments of theinvention.

As used herein, the singular forms “a”, “an”, and “the” include thecorresponding plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “an enzyme” includes aplurality of such enzymes, and a reference to “an amino acid” is areference to one or more amino acids. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any materials and methods similar orequivalent to those described herein can be used to practice or test thepresent invention, the preferred materials and methods are nowdescribed.

The term “alkyl” denotes straight chain, branched or cyclic alkyl.Examples of straight chain and branched alkyl include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl,isoamyl, sec-amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, hexyl,4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl,1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl,1,1,2-trimethylpropyl, and the like.

Examples of cyclic alkyl include mono- or polycyclic alkyl groups suchas cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The expression “optionally substituted” means that a group may or maynot be further substituted with one or more groups selected from alkyl,alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl,haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, carboxy, benzyloxy,haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl,nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, azido, amino,alkylamino, alkenylamino, alkynylamino, arylamino, benzylamino,acylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, acyloxy,aldehydo, alkylsulphonyl, arylsulphonyl, sulphonylamino,alkylsulphonylamino, arylsulphonylamino, alkylsulphonyloxy,arylsulphonyloxy, heterocyclyl, heterocycloxy, heterocyclylamino,haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy,carboaryloxy, mercapto, sulfonic acid, alkylthio, arylthio and acylthio.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacological and/or physiological effect. The effect may beprophylactic in terms of completely or partially preventing a diseaseinjury or a sign or symptom thereof, and/or may be therapeutic in termsof a partial or complete cure of a disease. “Treating” as used hereincovers any treatment of, or prevention of disease or injury in avertebrate, a mammal, particularly a human, and includes preventing thedisease from occurring in a subject who may be predisposed to thedisease, but has not yet been diagnosed as having it; inhibiting thedisease, ie., arresting its development; or relieving or amelioratingthe effects of the disease, ie., cause regression of the effects of thedisease.

As used herein, the term “therapeutically effective amount” means anamount of a compound of the present invention effective to yield adesired therapeutic response, for example to prevent or treat a diseasewhich is susceptible to treatment by administration of apharmaceutically-active agent.

The specific “therapeutically effective amount” will, of course, varywith such factors as the particular condition being treated, thephysical condition and clinical history of the subject, the type ofanimal being treated, the duration of the treatment, the nature ofconcurrent therapy (if any), and the specific formulations employed andthe structure of the compound or its derivatives.

As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent, excipient or vehicle fordelivering the compound of formula I and/or pharmaceutically-activeagent to the subject. The carrier may be liquid or solid, and isselected with the planned manner of administration in mind.

The compound of formula I and/or second pharmaceutically-active agentmay be administered orally, topically, or parenterally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles. The term parenteral asused herein includes subcutaneous, intravenous, intramuscular,intrathecal, intracranial, injection or infusion techniques.

The invention also provides suitable topical, oral, aerosol, andparenteral pharmaceutical formulations for use in the novel methods oftreatment of the present invention. The compounds of the invention maybe administered orally as tablets, aqueous or oily suspensions,lozenges, troches, powders, granules, emulsions, capsules, syrups orelixirs. The composition for oral use may contain one or more agentsselected from the group of sweetening agents, flavouring agents,colouring agents and preserving agents in order to producepharmaceutically elegant and palatable preparations. The tablets containthe active ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.

These excipients may be inert diluents, such as calcium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, such as corn starch or alginic acid; bindingagents, such as starch, gelatin or acacia; or lubricating agents, suchas magnesium stearate, stearic acid or talc. The tablets may beuncoated, or may be coated by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time-delaymaterial such as glyceryl monostearate or glyceryl distearate may beemployed. Coating may also be performed using techniques described inU.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

For in vivo application, the compounds of the invention or additionalpharmaceutically active agents can be administered parenterally byinjection or by gradual perfusion over time, independently or together.Administration may be intravenously, intra-arterial, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally. For invitro studies the compounds may be added or dissolved in an appropriatebiologically acceptable buffer and added to a cell or tissue.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers such as those based on Ringer's dextrose, and the like.Preservatives and other additives may also be present, such asanti-microbials, anti-oxidants, chelating agents, growth factors andinert gases and the like.

The invention includes various pharmaceutical compositions useful forameliorating disease or injury. The pharmaceutical compositionsaccording to one embodiment of the invention are prepared by bringing acompound of formula I, or an analogue, derivative or salt thereof, andone or more pharmaceutically-active agents or combinations of a compoundof formula I and one or more other pharmaceutically-active agents, intoa form suitable for administration to a subject, using carriers,excipients and additives or auxiliaries.

Carriers and formulations are described, for instance, in Remington'sPharmaceutical Sciences, 20th ed. Williams & Wilkins (2000) and TheBritish National Formulary 43rd ed. (British Medical Association andRoyal Pharmaceutical Society of Great Britain, 2002;http://bnf.rhn.net), the contents of which are hereby incorporated byreference. The pH and exact concentration of the various components ofthe pharmaceutical composition are adjusted according to routine skillsin the art. See Goodman and Gilman's The Pharmacological Basis forTherapeutics (7th ed., 1985).

The pharmaceutical compositions are preferably prepared and administeredin dosage units. Solid dosage units include tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the subject, different daily doses can be used.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the disease and the weight and general state of the subject.Typically, dosages used in vitro may provide useful guidance in theamounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of the cytotoxic side effects. Variousconsiderations are described in Langer, Science, 249: 1527, (1990).Formulations for oral use may be in the form of hard gelatin capsules,in which the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin. They may alsobe in the form of soft gelatin capsules, in which the active ingredientis mixed with water or an oil medium, such as peanut oil, liquidparaffin or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients may be suspending agents such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents, which may be

(a) a naturally occurring phosphatide such as lecithin;

(b) a condensation product of an alkylene oxide with a fatty acid, forexample, polyoxyethylene stearate;

(c) a condensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol;

(d) a condensation product of ethylene oxide with a partial esterderived from a fatty acid and hexitol such as polyoxyethylene sorbitolmonooleate, or

(e) a condensation product of ethylene oxide with a partial esterderived from fatty acids and hexitol anhydrides, for examplepolyoxyethylene sorbitan monooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known methods using suitable dispersing orwetting agents and suspending agents such as those mentioned above. Thesterile injectable preparation may also a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents which may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed, includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid may be used in the preparation of injectables.

Compounds of formula I may also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles, and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine, orphosphatidylcholines.

Dosage levels of the compound of formula I of the present invention willusually be of the order of about 0.5 mg to about 200 mg per kilogrambody weight, with a preferred dosage range between about 0.5 mg to about100 mg per kilogram body weight per day (from about 0.5 g to about 30 gper patient per day). The amount of active ingredient which may becombined with the carrier materials to produce a single dosage willvary, depending upon the host to be treated and the particular mode ofadministration. For example, a formulation intended for oraladministration to humans may contain about 5 mg to 1 g of an activecompound with an appropriate and convenient amount of carrier material,which may vary from about 5 to 95 percent of the total composition.Dosage unit forms will generally contain between from about 5 mg to 500mg of active ingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

In addition, some of the compounds of the invention may form solvateswith water or common organic solvents. Such solvates are encompassedwithin the scope of the invention.

The compounds of the invention may additionally be combined with othercompounds to provide an operative combination. It is intended to includeany chemically compatible combination of pharmaceutically-active agents,as long as the combination does not eliminate the activity of thecompound of formula I of this invention.

The methods of this invention may involve the administration of acompound of formula I, prior to, together with, or subsequent to theadministration of a second pharmaceutically-active agent; or theadministration of a combination of a compound of formula I and a secondpharmaceutically-active agent.

In certain preferred embodiments, the invention provides new quaternaryimidazolium derivatives, namely:

in which R¹ and R² are methyl or ethyl alkyl groups, and X⁻ is anorganic or non-organic acid anion. Particularly preferred compounds arethose in which:R¹ and R² are CH₃, X⁻ is benzenesulphonate (1);R¹ is CH₃, R² is C₂H₅, X⁻ is benzenesulphonate (2);R¹ and R² are C₂H₅, X⁻ is benzenesulphonate (3);R¹ is CH₃, R² is C₂H₅, X⁻ is Cl (4);R¹ is CH₃, R² is C₂H₅, X⁻ is benzoate (5);R¹ is CH₃, R² is C₂H₅, X⁻ is salicylate (6);R¹ is CH₃, R² is C₂H₅, X⁻ is dihydroxybenzoate (gentisate) (7).

Compounds 1-3 can be synthesised by quaternization of 1-alkyl-4,5-bis(N-methylcarbamoyl)imidazolium on heating with an alkyl ester ofbenzenesulphonate, in, accordance with the following reaction:

Compounds 4-7 can be synthesised by replacing the anion in1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazolium benzenesulfonate(2) by ion-exchange, in accordance with the following scheme:

The compounds of the invention are readily soluble in water, stable, andnon-toxic.

Because inflammation is a complex process consisting of several stagesand involving inflammatory mediators, a uniform model of inflammation isnot feasible. The effect of 1,3-dialkyl-4,5-bis (optionallyN-substituted carbamoyl)imidazolium salts was assessed on models whichare widely used in researching the anti-inflammatory properties ofmedications. The effect of the compounds of the invention was assessedwith regard to both preventive and therapeutic treatments.

Experimental inflammation in mice and rats was induced by variousmethods. As models of acute inflammation we used induction of paw oedemain response to the administration of concanavalin A (Con A; Serva),carrageenin (Serva) or bradykinin (Sigma). As a model of chronicinflammation we used cotton wool-induced granuloma. Our resultsdemonstrated that the compounds prevent acute inflammation caused byvarious inflammatory agents (prophylactic effect) and reduce thedevelopment of chronic inflammation (treatment effect), to a degreecomparable to the effect of well-known anti-inflammatory medicationssuch as non-steroidal anti-inflammatory agents.

The acuteness of the inflammation was assessed 30 minutes after theinjection of bradykinin and 3 hours after injection of carrageeninrespectively, by measuring the change in paw volume. Theanti-inflammatory effect was assessed by the reduction of swelling wasexpressed in % relative to controls, or the reaction index of the anklejoint inflammation was assessed by weighing the experiment and controlpaws. The test substances were administered orally via an intra-gastrictube one hour before carrageenin or bradykinin was administered. Ascomparative medications, the following non-steroidal anti-inflammatoryagents were also administered orally: butadione 60 mg/kg, ibuprofen 48mg/kg and paramidine 50 mg/kg. These dosages correspond to ED₅₀ valuesfrom the literature.

For inflammation induced by carrageenin or bradykinin, the testsubstances were injected 4 times, daily for 3 days and 1 hourimmediately before the administration of the inflammatory agent. Forconcanavalin A, the test substances were administered once, one hourprior to administration of Con A. The results obtained confirm that thecompounds of the invention can reduce acute inflammation caused byconcanavalin A, carrageenin or bradykinin in intact animals, both whenadministered in a single dose or continuously, and in adrenalectomizedas well as in intact animals.

It is well known that when medications are applied to the skin, theirphysical and chemical characteristics, such as ionisation ability (pKa)at the pH of the structural elements of the skin and their lipophilicity(logP) affect the rate at which the active agents are released from themedication and absorbed into the skin. Because the compounds of theinvention have low lipophilicity (logP approximately −0.95), theypenetrate biological membranes poorly, thus helping to prolong contactwith the damaged area. Both these factors prolong the effect of thecompounds, increasing their efficiency when applied locally. Therefore,these agents are suitable for topical application, for example as anointment, solution, or finely dispersed powder.

The invention will now be described in detail by way of reference onlyto the following non-limiting examples and drawings.

EXAMPLE 1 1,3-dimethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumbenzenesulfonate (1)

1 g of 1-methyl-4,5-bis(N-methylcarbamoyl)imidazolium[bis(N-methylamide) 1-methylimidazolium-4,5-dicarboxylic acid] washeated in 5 ml of methyl ether of benzenesulphonic acid at 120° C. for 3hours. The reaction mixture was diluted with an appropriate solvent,such as water-free diethyl ether or water-free acetone, and the residuewas filtered. The yield was 1.5g (85.7%). The melting point was 159-161°C. (n-butanol n-heptane). The results of elemental analysis and NMRspectroscopy are set out in Tables 1 and 2 respectively.

EXAMPLE 2 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumbenzenesulfonate (2)

1 g of 1-methyl-4,5-bis(N-methylcarbamoyl)-imidazolium[bis(N-methylamide) 1-ethylimidazolium-4,5-dicarboxylic acid] was heatedin 5 ml of benzenesulphonic acid methyl ether at 120° C. for 3 hours.The reaction mixture was diluted by an appropriate solvent, such aswater-free diethyl ether or water-free acetone, and the residue wasfiltered. The yield was 1.5 g (82.5%). The melting point was 133-135° C.(n-butanol n-heptane). The results of elemental analysis and NMRspectroscopy are set out in Tables 1 and 2 respectively.

EXAMPLE 3 1,3-diethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumbenzenesulfonate (3)

1 g of 1-ethyl-4,5-bis(N-methylcarbamoyl)-imidazolium[bis(N-methylamide) 1-ethylimidazolium-4,5-dicarboxylic acid] was heatedin 5 ml of benzenesulphonic acid ethyl ether at 120° C. for 3 hours. Thereaction mixture was diluted by an appropriate solvent, such aswater-free diethyl ether or water-free acetone, and the residue wasfiltered. The yield was 1.4 g (74.2%). The melting point was 108-111° C.(n-butanol n-heptane). The results of elemental analysis and NMRspectroscopy are set out in Tables 1 and 2 respectively.

EXAMPLE 4 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumchloride (4)

1 g of 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumbenzenesulfonate (2) was dissolved in 20 ml of distilled water, and thesolution was then heated with the ion-exchange resin Amberlite IRA-410(Cl⁻-form, 40 ml, eluent water). 600 ml of eluent was collected, and thewater was evaporated under vacuum. The residue was dried under vacuumover P₂O₅ at room temperature for 12-18 hours. Compound (4) was obtainedas a white hygroscopic powder. The yield was 0.68 g (98%). The meltingpoint was 192-194° C. The results of elemental analysis and NMRspectroscopy are set out in Tables 1 and 2 respectively.

EXAMPLE 5 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumbenzoate (5)

1 g of 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumbenzenesulfonate (2) was dissolved in 20 ml of distilled water, and thesolution was then heated with the ion-exchange resin Amberlite IRA-410(C₆H₅COO⁻-form, 40 ml, water eluent). 600 ml of eluent was collected,and the water was evaporated under vacuum. The residue was dried undervacuum above P₂O₅ at room temperature for 12-18 hours. Compound (5) wasobtained as a white hygroscopic powder. The yield was 0.89 g (97%). Themelting point was 140-145° C. The results of elemental analysis and NMRspectroscopy are set out in Tables 1 and 2 respectively.

EXAMPLE 6 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumsalicylate (6)

6 g of 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazoliumbenzenesulfonate (2) were dissolved in 200 ml of distilled water. Thesolution was then heated with ion-exchange resin Amberlite IRA-410 10(OH⁻-form), and the eluent was collected in a 2-litre beaker containing3.5 g (0.024 mole) of salicylic acid per 15 ml of water. The volume ofthe eluent was 1.5 litres. The undissolved salicylic acid was filtered,and the dihydrate of compound (6) was extracted by lyophilization togive a light yellow hygroscopic powder. The yield was 5.85 g (98%). Themelting point was 109-110° C. The results of elemental analysis and NMRspectroscopy are set out in Tables 1 and 2 respectively.

EXAMPLE 7 1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazolium2,4-dihydroxybenzoate (7)

6 g (0.016 mole) of1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)-imidazolium benzenesulfonate(2) were dissolved in 200 ml of distilled water. The solution was thenheated with ion-exchange resin Amberlite IRA-410 10 (OH⁻-form), and theeluent was collected in a 2-litre beaker containing 3.85 g (0.025 mole)of 2.5 dihydroxybenzoic acid per 15 ml of water. The volume of theeluent was 1.5 litres. The undissolved acid was filtered, and compound(7) was extracted by lyophilization to give a light yellow hygroscopicpowder. The yield was 5.76 g (97%). The melting point was 168-170° C.The results of elemental analysis and NMR spectroscopy are set out inTables 1 and 2 respectively.

EXAMPLE 8 Alternative synthesis of Compounds (6) and (7)

Compounds (6) and (7) can also be obtained by heating the solution ofcompound (2) with ion-exchange resin containing salicylic or 2.5dihydroxybenzoic acid, similarly to the method used to produce compounds(4) and (5), with the final products being isolated by lyophilization.TABLE 1 Results of elemental analysis of compounds I-VII Com- MolecularFound Calculated pound formula C H N C H N 1 C₁₅H₂₀N₄O₅S 48.95 5.6014.63 48.90 5.47 15.21 48.48 5.47 14.87 2 C₁₆H₂₂N₄O₅S 50.81 5.84 14.4150.25 5.80 14.65 50.81 6.13 14.65 3 C₁₇H₂₄N₄O₅S 51.18 6.07 14.13 51.506.10 14.13 51.23 6.11 14.33 4 C₁₀H₁₇N₄O₂Cl 46.13 6.57 21.47 46.07 6.5721.29 46.35 6.31 21.06 5 C₁₇H₂₂N₄O₄ 58.75 6.39 15.94 58.93 6.41 16.1859.08 6.77 16.09 6 C₁₇H₂₂N₄O₅ 2H₂O 51.71 6.40 13.94 51.25 6.58 14.0651.55 6.35 14.21 7 C₁₇H₂₂N₄O₆ 14.93 14.81 15.01

TABLE 2 Results of NMR analysis of compounds 1-7(DMSO-d6) Com- pound δ,ppm. 1 2.78 s[3H, (NH)—CH₃], 2.80 s[3H, (NH)—CH₃], 3.87 s[6H,N1(3)—CH₃], 7.31-7.60[5H, arom H], 8.77-8.78[2H, NH], 9.26 s[1H, (C2)—H]2 1.37 t[3H, CH₂—CH₃, J=7Hz], 2.79 s[3H, NH—CH₃], 2.80 s[3H, (NH)—CH₃],3.87 s[3H, N1—CH₃], 4.28 q[2H, CH₂, J=7Hz], 7.32-7.60[5H, arom H],8.73-8.82[2H, NH], 9.35 s[1H, (C2)—H] 3 1.38 t[6H, 2(CH₂)—CH₃, J=7Hz],2.80 s[6H, 2(NH)—CH₃], 4.30 q[2H, 2(CH₃)—CH₂, J=7Hz], 7.31-7.59[5H, aromH], 8.80[2H, 2NH], 9.41 s[1H, (C2)—H] 4 1.38 t[3H, CH₂—CH₃, J=7Hz],2.78-2.79[6H, (NH)—CH₃], 3.90 s[3H, N1—CH₃], 4.30 q[2H, CH₂, J=7Hz],9.35 s[1H, NH], 9.42 s[1H, NH], 9.64 s[1H, (C2)—H] 5 1.31 t[3H, CH₂—CH₃,J=7Hz], 2.81 s[6H, 2 NH—CH₃], 3.79 s[3H, N1—CH₃], 4.20 q[2H, CH₂,J=7Hz], 7.29-7.78 m[5H, arom H], 9.42-10.1[3H, 2 NH, (C2)—H] 6 1.37t[3H, CH₂—CH₃, J=7Hz], 2.81 s[6H, 2 NH—CH₃], 3.87 s[3H, N1—CH₃], 4.30q[2H, CH₂, J=7Hz], 6.63-7.71[4H, arom H], 8.94 s[1H, NH], 9.00 s[1H,NH], 9.40 s[1H, (C2)—H] 7 1.37 t[3H, CH₂—CH₃, J=7Hz], 2.80 s[6H, 2NH—CH₃], 3.86 s[3H, N1—CH₃], 4.28 q[2H, CH₂, J=7Hz], 6.45-7.15[3H, aromH], 8.5 s[1H], 9.02-9.08 s[2H, 2NH], 9.40 s[1H, (C2)—H]

EXAMPLE 9 Effect of 1,3-dialkyl-4,5-bis (N-methylcarbamoyl)imidazoliumsalts on acute Con A-induced inflammation

1,3-dialkyl-4,5-bis (N-methylcarbamoyl)imidazolium salts at doses of 10and 50 mg/kg were injected intra-peritoneally into male CBA mice,weighing between 18 and 20 g, one hour before the injection ofconcanavalin A (con A). Saline solution was similarly administered tothe control animals. There were 10 mice in each group. After one hour,con A at a dose of 100 mkg/20 g of body weight was administered bysub-plantar injection into the plantar aponeurosis of the left paws ofmice of both the experimental and control groups. The same amount ofsaline splution was injected into the contralateral paws. After onehour, the mice were killed and the experimental and control paws wereweighed to assess the reaction index of the inflammation in theankle-joint (Liubimov et al, 1999).

We found that the maximum swelling of ankle joints was observed 1 hourafter the sub-plantar administration of Con A. Intraperitonealadministration of 1,3-dialkyl-4,5-bis(N-methylcarbamoyl)-imidazoliumsalts at a dose of 10 to 50 mg/kg one hour before the induction of theoedema reduced the intensity of the inflammatory process. These resultsare summarised in Table 3. TABLE 3 Effect of 1,3-dialkyl-4,5-bis(N-methylcarbamoyl)-imidazolium salts on acute Con A-inducedinflammation in mice Medication Reaction index (%) Saline solution 20.2± 3.8  Compound (1)10 mg/kg 14.6 ± 3.5  Compound (1)50 mg/kg  5.3 ±2.0** Compound (2)10 mg/kg 10.4 ± 2.0*  Compound (2)50 mg/kg 8.4 ± 0.9*Compound (3)10 mg/kg 9.5 ± 2.5* Compound (3)50 mg/kg 9.5 ± 2.0* Compound(4)10 mg/kg 14.1 ± 2.0  Compound (4)50 mg/kg 12.4 ± 0.4*  Compound (5)50mg/kg 7.2 ± 1.8**significant difference between experimental group and control (Con A) -P < 0.001;**difference between the effect of compound (1)(50 mg/kg) and controls.

EXAMPLE 10 Comparison of the anti-inflammatory effect of Compound 2 andCompound 7

The anti-inflammatory effect of Compound 2 and Compound 7 was comparedin male mice weighing 18-22 g, using the model described in Example 9.The ED₅₀, i.e. the dose reducing paw ooedema by 50% one hour afterinjection of Concanavalin A, was calculated. The results are shown inTable 4, and indicate that ED₅₀ for Compound 2 was 33 mg/kg, and ED₅₀for Compound 7 was 52 mg/kg. TABLE 4 Comparison of anti-inflammatoryeffect of compound 2 and compound 7 Reaction Paw oedema Group of animalsIndex reduction n = 10 (%) (%) Saline solution + Con A 16.0 0 Compound 210 mg/kg + Con A 11.1 ± 3.5  33 Compound 2 19 mg/kg + Con A 5.4 ± 2.0 66(0.005M) Compound 2 38 mg/kg + Con A 7.7 ± 1.8 52 (0.01M) Compound 2 57mg/kg + Con A 5.3 ± 2.0 66 (0.015M) Compound 2 76 mg/kg + Con A 3.3 ±2.0 77 (0.02M) Compound 7 16.4 mg/kg + Con A 6.73 ± 2.2  60 (0.005M)Compound 7 32 mg/kg + Con A 9.93 ± 2.8  13 (0.01M) Compound 7 49 mg/kg +Con A 7.2 ± 1.8 45 (0.015M) Compound 7 65.6 mg/kg + Con A 4.9 ± 1.7 70(0.02M)

EXAMPLE 11 Effect of Compound (2) on carrageenin-induced inflammation inintact and adrenalectomized animals

An acute inflammatory oedema was induced in intact and adrenalectomizedmale rats weighing 180-200 g by sub-plantar injection of 0.1 ml of 1%carrageenin solution into the left paw (Winter et al., 1962). The samevolume of saline solution was injected into the contralateral paw.Compound (2) at doses of 50 and 100 mg/kg and ibuprofen as a comparativemedication at a dose of 48 mg/kg, corresponding to the literature ED₅₀value, were administered orally into the stomach via an intra-gastrictube 4 times, daily each day for 3 days and one hour beforeadministration of carrageenin. There were 10 rats in each group.

The degree of carrageenin-induced inflammation of the ankle joints wasassessed at 3 hours after the induction of the inflammation, on thebasis of the change in weight of the experimental and control paws(reaction index). The results are summarised in Table 5, and show thatcompound 2 reproducibly reduced the inflammation in both intact andadrenalectomized animals. TABLE 5 Effect of prolonged administration ofcompound (1) on the stage of acute inflammation in rats caused bycarrageenin Reaction index Inhibition of Group (%) oedema(%) Intactanimals + saline 43.04 ± 3.58 0 solution + carrageenin Intact animals +compound 29.96 ± 2.30*,** 30.41 (2)50 mg/kg 4 days + carrageeninAdrenalectomized animals + compound 31.04 ± 2.49*,** 27.88 (2)50 mg/kg 4days + carrageenin Intact animals + compound 22.63 ± 2.86*,** 47.43(2)100 mg/kg 4 days + carrageenin Adrenalectomized animals + compound25.63 ± 2.86*,** 44.43 (2)100 mg/kg 4 days + carrrageenin Ibuprofen 48mg/kg 4 days + 15.88 ± 1.88* 63.39 carrageenin Adrenalectomizedanimals + ibuprofen 18.12 ± 1.82* 57.90 48 mg/kg orally 4 days +carrageenin*significant difference between the experiment groups and controls, P <0:001;**significant difference between the effect of compound (I) andcontrols.

EXAMPLE 12 Effect of Compound (2) on bradykinin-induced inflammation inintact animals

Acute inflammation was induced by sub-plantar injection of 0.1 ml of0.01% of bradykinin in saline solution into the left paws of male ratsweighing 180-200 g. The acuteness of the inflammation was assessed 30minutes after injection of bradykinin by the change in the paw volume.The anti-inflammatory effect, assessed by the reduction of swelling, wasexpressed as a percentage of the initial paw weight compared tocontrols. The test substances were administered orally via anintra-gastric tube for three days and at one hour before administrationof bradykinin. The following agents were used for comparison:

-   -   Control: saline alone    -   Butadione: 60 mg/kg: (anti-inflammatory agent)    -   Paramidine: 50 mg/kg (pyridinole carbamate; Buclome (Takeda);        specific anti-bradykinin agent).        These were administered orally to rats for 3 days and at one        hour before the injection of the agent. There were 12 rats in        each group. The doses of butadione and paramidine correspond to        literature ED₅₀ values.

Compound (2) at a dose of 50 mg/kg resulted in an obvious lessening ofthe swelling reaction caused by bradykinin. This effect was equal to theeffect of the specific anti-bradykinin activity of paramidin, andsimilar to that of butadione. The results are summarised in Table 6.TABLE 6 Effect of compound (2) on acute bradykinin-induced inflammationin rats Paw volume (ml of water reduction) 30 minutes Increase after inpaw Swelling administration volume inhibition Medication initial ofbradykinin (ml) (%) Bradykinin 1.23 ± 0.043 1.92 ± 0.070 0.69 0 Compound1.39 ± 0.040 1.80 ± 0.060 0.41 40.58 (2) 20 mg/kg Compound 1.35 ± 0.038 1.69 ± 0.060* 0.34 50.72 (2) 50 mg/kg Paramidine 1.27 ± 0.038  1.61 ±0.051* 0.34 50.72 50 mg/kg Butadione 1.33 ± 0.061 1.60 ± 0.07* 0.2760.87 60 mg/kg*significant difference between butadione, paramidine, compound (2) andcontrols - P < 0:001.

EXAMPLE 13 Effect of compound (2) on chronic proliferative inflammation(granuloma)

We investigated the ability of compound (2) to reduce the formation ofgranulation tissue in the inflammatory focus which develops at the siteat which a non-sterile cotton-wool roll weighing 40 mg is implantedsubcutaneously (Swingle and Shideman, 1972). Compound (2) wasadministered orally to male rats weighing 130-150 g at a dose of 100mg/kg for 6 days before the cotton-wool roll was implanted, and then for6 days at the same dose while the granuloma developed. Anti-inflammatoryagents, such as ibuprofen and butadione, were used for comparison, usingthe same procedure. There were 10 rats in each group.

After 12 days the experiment was terminated, and the granulomas wereexcised, weighed, and the wet weight of the granulation tissue wasmeasured. The dry mass of the granuloma was measured after drying for 24hours at 70° C., and the exudative and proliferative stages of thegranulomas were calculated. Statistical analysis was performed using theStudent's parametric test. The results are summarised in Table 7, andshow that compound (2) is able to inhibit granuloma formation to anextent similar to that of conventional anti-inflammatory agents. TABLE 7Effect of compound (2) on the development of experimental granuloma inrats Granuloma weight (mg) Group Fresh weight Dry weight Controls 1375.8± 61.85   249.6 ± 13.27 (0.5 ml starch) Exudative stage Proliferative1126.2  stages 209.6 Compound (2) 956.29 ± 115.5*  180.71 ± 14.16* (100mg/kg) Exudative stage Proliferative  775.58 stages  140.71 Ibuprofen(48 mg/kg) 814.0 ± 63.72**  143.2 ± 9.83** Exudative stage Proliferative670.8 stages 103.2 Butadione (60 mg/kg 828.0 ± 76.68**  145.4 ± 7.45**Exudative stage Proliferative 682.6 stages 105.4*significant difference between the second experimental group andcontrols (P < 0.01);**significant difference between the third experimental group andcontrols; significant difference between the fourth group and controls(P < 0.001).

EXAMPLE 14 Effect of topically-applied Compound 2 on wound healing

To heal successfully, wounds should be treated with regard to specificfeatures of the various stages of healing process of the wound (Kuzin etal, 1981). The wound-healing effect of salts of N-substituted1,3-dialkyl-4,5-bis(carbamoyl)imidazolium derivatives was studied in amodel of aseptic full-thickness skin wounds in rats (Zapadnyuk et al,1983). The effect of 10%1-ethyl-3-methyl-4,5-bis(N-methylcarbamoyl)imidazolium benzensulfonatein a lanolin ointment base was compared to that of Solcoseryl gel andSpasatel balm.

Statistical analysis of the data was performed using standard methodsinvolving the Student's parametric test, based on the programmedeveloped in the Department of Neuropharmacology of the Institute ofExperimental Medicine of the Russian Academy of Medical Sciences.Healing of a full-thickness wound was assessed on the basis of a set ofmorphological criteria characterising the quality of the regenerationprocess.

Full-thickness wounds are wounds in which all three layers of the skin,ie. the epidermis, dermis and the subdermis are damaged. Reparative andregenerative tissue processes affect each of the three layers. Healingoccurs by so-called “primary intention” if the wound is aseptic or isthoroughly cleaned early in the process; regenerative processes in theepithelium occur in tandem and are complete by the fifteenth day, withformation of granulation tissue and proliferation and differentiation ofregenerative tissues. “Secondary intention” healing is healing in thepresence of infection or after loss of a large area of tissue, forexample following major trauma or burn. Secondary intention healing isalways accompanied by scar formation.

Aseptic full-thickness skin wounds were created in rats under lightether anaesthesia. At a site on the animal's back where the animal couldnot lick the wound, the skin was shaved, and a circular piece of skinapproximately 2.5 cm² area was excised with scissors. Following recoveryfrom anaesthesia the animals were kept in separate cages, with normalfood and water ad libitum. A total of 100 rats weighing from 200 to 250g were used, with 20 rats in each group. The groups were as follows:

1. Untreated control (self-healing)

2. Placebo control (lanolin ointment base only)

3. Test (10% Compound 2 in lanolin ointment base)

4. Comparative Group A: Solcoseryl gel (Solco, Switzerland)

5. Comparative Group B: Spasatel balm (AOE Effect, Ukraine).

Treatment was performed 24 hours after the wounding by applying 200 g ofointment once a day in a proportion of 80 mg of ointment per 1 cm² ofthe wound area (8 mg of substance per 1 cm²) and then daily untilhealing was complete.

Solcoseryl is widely used in Russia as a wound healing medication; thiscomposition contains deproteinised haemodialysate of male calf blood. Ithas been shown to induce healing by secondary intention. Spasatel is amulti-component balsam containing sea buckthorn oil, naphthalene,beeswax, essential oils derived from various plants, and vitamins. Thiscomposition induces healing by secondary intention, and often leavesresidual scarring.

Animals were examined at 5, 10, 15 and 20 days for the followingparameters; wound size and wound healing time; weight gain, peripheralblood indicators (total leucocyte count, differential leucocyte count,and erythrocyte sedimentation rate); and biochemical serum indicators(total protein, albumin, globulin, and globulin fractions); andmacroscopic and microscopic assessment of wounds. The average completewound healing period was 16.8±1.9 days for Compound 2, compared to23.5±2.2 days for Spasatel and 24.0±2.4 days for Solcoseryl. Thus thecompound of the invention was significantly more effective than eitherof the prior art agents.

For measurement of wound size, a sterile object plate was laid over thewound, and its contour was outlined with a marker. The wounds werephotographed, the wound outlines were scanned, and their area wascalculated using an in-house computer program.

The results of the re-measurement of the wound area illustrate thedynamics of regeneration processes of both the epithelium covering thewound and the underlying granulation tissue. The average area figures incontrol and test groups were compared. A comparative analysis of thewound healing dynamics indicated that the wound area for the animalstreated with 10% Compound 2 ointment was 0.4% on the fifteenth day,compared to 10.4% for Solcoseryl, 11% for Spasatel and 24.1% forplacebo.

The results are presented in Tables 8 to 10. The most marked woundhealing effect was observed with Compound 2 ointment; the differencefrom the controls and from the comparison groups was significant(p<0.05).

Healing in response to treatment with Compound 2 takes place by primaryintention, in which the wound is rapidly and fully cleaned, andregeneration processes in the epithelium-connective tissue system, whichare synchronised and completed by the 15th day, proceed without anyevidence of pathological regeneration or differentiation of connectivetissue. TABLE 8 Duration of wound healing Epithelialization Nos. Groupof animals (duration in days) 1. Control (self-healing) 29.2 ± 1.1 2.Placebo¹ 24.5 ± 1.1* 3. Compound 2² (10% 16.8 ± 1.9*^(.)** ointment) 4.Solcoseryl gel 24.0 ± 2.4* 5. Spasatel balm 23.5 ± 2.3*¹ointment base - lanolin²1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzensulfonate*significant difference compared to control (P < 0.05)**significant difference compared to placebo (P < 0.05)

TABLE 9 WOUND HEALING DYNAMICS Wound area (% of area before treatment)Before Animal Group treatment 5^(th) day 10^(th) day 15^(th) day 20^(th)day 1. Placebo¹ 100.0 106.9 43.8 24.1 11.6 2. Test compound² 100.0 84.08.1 0.4 0.1 (10% ointment) 3. Solcoseryl 100.0 89.2 22.6 10.4 3.8 4.Spasatel 100.0 89.6 23.8 11.0 5.8¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (compound 2)

TABLE 10 WOUND HEALING PERIOD Animal group Epithelialization period 1.Control 29.2 ± 1.1 (untreated) 2. Placebo¹ 24.5 ± 1.1 3. Test compound²16.8 ± 1.9** (10% ointment) 4. Solcoseryl 24.0 ± 2.4 5. Spasatel 23.5 ±2.2*differences are significant as compared to the control group(self-healing), P < 0.05**differences are significant as compared to the control group(placebo), P < 0.05¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (compound 2)

Body weight was checked once a week before feeding the animals. Theresults are presented in table 11. Daily observations indicated that astatistically significant weight gain was observed only in the animalswho were treated with Compound 2. On the twentieth day, in the testgroup this indicator was 19%, whereas in the control (untreated andplacebo) groups it was 10-12%. These results confirm the positive effectof the test compound on wound repair and regeneration processes. TABLE11 Rat body weight changes during full-thickness wound treatment Beforetreatment 5^(th) day 10^(th) day 15^(th) day 20^(th) day Animal Group g% g % g % g % g % 1. Control 272 ± 12.6 100 279 ± 10.5 102.6 279 ± 8.4102.6 294 ± 11.6 108.1   301 ± 12.6 110.7 (untreated) 2. Placebo¹ 229 ±13.7 100 240 ± 16.8 104.8  245 ± 12.6 107.0 252.5 ± 12.6   110.3 257.5 ±12.6 112.4 3. Test compound² 226 ± 8.4  100 236 ± 8.4  104.4 246 ± 1.1108.8 253.3 ± 2.1   112.1 268.0 ± 4.2  118.6 (10% ointment) 4.Solcoseryl 235 ± 6.3  100 250 ± 6.3  106.4 264 ± 8.4 112.3 272 ± 10.5115.7 279.0 ± 8.4  118.7 5. Spasatel 230 ± 8.4  100 244 ± 10.5 106.1 250 ± 10.5 108.7 255 ± 12.6 110.9 258.0 ± 12.6 112.2¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (Compound 2)

Peripheral blood indicators (leucocytes, leucocyte formula, erythrocytesedimentation rate) of the rats from the test and control groups weremeasured before the animals were wounded, and then checked on the fifth,tenth and fifteenth days of the experiment. The results of theseexperiments are presented in Tables 12, 13, and 14. No reproduciblechanges compared to the blood characteristics of the intact animalsprior to wounding were identified from the haematological indicators ofrats treated with Compound 2. TABLE 12 Rat peripheral blood indicatorsduring full-thickness skin wound treatment - day 5 Leucocyte count (%)Animal Leucocytes Band Segmented group ESR mm/h 10⁹/l neutrophilsneutrophils eosinophils basophils monocytes lymphocytes 1 Intact 2.3 ±1.3 7.2 ± 1.3 1.3 ± 0.3 30.0 ± 5.1 1.7 ± 0.3 0 3.7 ± 0.7 63.3 ± 6.2 5days 2 Control 1.8 ± 0.4 5.3 ± 0.2 1.2 ± 0.2 43.2 ± 3.0 2.0 ± 0.4 0.4 ±0.2 3.6 ± 0.9 49.6 ± 3.7 (untreated) 3 Placebo¹ 1.8 ± 0.4 5.5 ± 0.3 1.2± 0.2 37.8 ± 3.9 2.8 ± 0.6 0.4 ± 0.2 5.0 ± 1.1 52.8 ± 3.2 4 Test 2.2 ±0.6 6.6 ± 0.9 1.4 ± 0.4 35.2 ± 4.4 2.6 ± 0.6 0 8.2 ± 0.8 52.6 ± 4.5compound² (10% ointment) 5 Solcoseryl 1.0 ± 0   5.8 ± 0.4 1.2 ± 0.2 40.2± 2.5 3.2 ± 0.7 0.4 ± 0.2 4.4 ± 0.8 51.0 ± 3.3 6 Spasatel 1.8 ± 0.4 5.2± 0.4 1.0 ± 0   39.4 ± 3.0 3.6 ± 0.5 0 5.0 ± 1.2 50.8 ± 2.7¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (Compound 2)

TABLE 13 Rat peripheral blood indicators during full-thickness skinwound treatment - day 10 Leucocyte count (%) Animal Leucocytes BandSegmented group ESR mm/h 10⁹/l neutrophils neutrophils eosinophilsbasophils monocytes lymphocytes 10 days 1 Control 1.2 ± 0.2 8.6 ± 1.03.0 ± 0.5 23.5 ± 5.2 2.7 ± 0.5 0.4 ± 0.2 3.4 ± 0.6 67.0 ± 5.9(untreated) 2 Placebo¹ 1.3 ± 0.3 7.2 ± 1.1 1.3 ± 0.3 39.3 ± 1.3 3.3 ±1.0 0 6.0 ± 0.7 50.3 ± 1.7 3 Test 1.0 ± 0   7.9 ± 0.6 1.2 ± 0.2 29.6 ±2.2 2.4 ± 0.5 0.6 ± 0.2 2.4 ± 0.5 63.8 ± 1.9 compound² (10% ointment) 4Solcoseryl 1.6 ± 0.4 9.2 ± 0.8 1.8 ± 0.4 29.6 ± 2.5 3.2 ± 0.6 0.2 ± 0.22.8 ± 0.6 62.4 ± 2.5 5 Spasatel 1.2 ± 0.2 9.4 ± 0.8 1.4 ± 0.2 40.0 ± 3.02.0 ± 0.6 0.2 ± 0.2 1.8 ± 0.4 54.6 ± 2.3¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (Compound 2)

TABLE 14 RAT PERIPHERAL BLOOD INDICATORS DURING FULL-THICKNESS SKINWOUND TREATMENT - DAY 15 European Search Leucocyte count (%) AnimalReport Leucocytes Band Segmented group mm/h 10⁹/1 neutrophilsneutrophils eosinophils basophils monocytes lymphocytes 15 days 1Control 1.8 ± 0.4 10.1 ± 2.3  1.8 ± 0.4 35.4 ± 5.7 3.6 ± 1.7 0.2 ± 0.25.4 ± 1.7 53.8 ± 5.8 (untreated) 2 Placebo¹ 1.4 ± 0.2 9.4 ± 1.4 1.4 ±0.2 29.4 ± 2.6 2.2 ± 0.6 0.4 ± 0.2 4.8 ± 0.6 62.0 ± 2.3 3 Test 1.4 ± 0.211.1 ± 1.6  1.2 ± 0.2 21.0 ± 3.1 2.8 ± 0.2 0.4 ± 0.2 4.0 ± 0.7 70.6 ±3.4 compound² (10% ointment) 4 Solcoseryl 1.6 ± 0.2 7.8 ± 1.2 1.2 ± 0.231.8 ± 4.9 5.6 ± 1.3 0.4 ± 0.2 4.2 ± 0.9 56.0 ± 5.3 5 Spasatel 1.4 ± 0.26.6 ± 0.7 1.0 ± 0   34.0 ± 2.4 3.0 ± 0.9 0.6 ± 0.4 6.4 ± 1.2 55.0 ± 1.9¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (Compound 2)

Total protein in the blood of test and control animals was determinedusing the biuret method, and protein fractions (albumins and globulins:α₁, α₂, β and γ) were measured electrophoretically prior to theexperiment and then on the fifth, tenth and fifteenth days. The datapresented in tables 15, 16, and 17 show that on the fifth day all groupsof animals demonstrated a decrease in albumin and increase in globulinfractions in their blood as a result of inflammation. These valuesreturned to normal levels at days 10 and 15. These changes in theprotein parameters reflect general patterns in response to skin damage.TABLE 15 Total serum protein and protein fractions - day 5 Animal TotalGlobulins Group Protein Albumin % Globulin % á₁ á₂ â ã A/G 1 Intact65.57 ± 1.15 44.8 ± 0.7 55.2 ± 0.7 12.1 ± 1.4  9.0 ± 1.5  21.4 ± 1.9912.7 ± 1.2 0.82 ± 0.02 5 days 2 Control 56.80 ± 1.72 39.2 ± 3.6 60.8 ±3.6 19.4 ± 0.9 10.9 ± 1.1  16.5 ± 0.96 14.1 ± 3.1 0.67 ± 0.09(untreated) 3 Placebo¹ 59.34 ± 0.89 41.8 ± 1.4 58.2 ± 1.4 17.1 ± 1.513.3 ± 0.6 17.6 ± 2.3 10.1 ± 1.2 0.72 ± 0.04 4 Test 57.45 ± 0.64 37.2 ±2.5 62.8 ± 2.5 16.8 ± 0.4 16.0 ± 1.5 18.3 ± 1.4 11.3 ± 0.7 0.60 ± 0.07compound² (10% ointment) 5 Solcoseryl 61.51 ± 2.18 37.3 ± 2.0 62.7 ± 2.017.0 ± 1.2 15.7 ± 1.5 21.5 ± 1.6  8.5 ± 1.3 0.60 ± 0.05 6 Spasatel 59.54± 2.05 41.0 ± 2.3 59.0 ± 2.3 19.6 ± 1.5 14.0 ± 1.3 16.7 ± 1.4  8.7 ± 0.50.71 ± 0.07¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (Compound 2)

TABLE 16 Total serum protein and protein fractions - day 10 Animal TotalGlobulins Group Protein Albumin % Globulin % á₁ á₂ â ã A/G 10 days 1Control 56.40 ± 0.98 43.5 ± 0.8 56.5 ± 0.8 11.9 ± 0.8 9.5 ± 0.9 21.0 ±0.8 14.2 ± 0.6 0.77 ± 0.03 (untreated) 2 Placebo¹ 66.88 ± 1.07 50.0 ±1.9 50.0 ± 1.9 12.5 ± 0.3 8.3 ± 0.3 18.2 ± 1.6 11.0 ± 0.8 1.01 ± 0.08 3Test 64.77 ± 1.44 42.9 ± 0.9 57.1 ± 0.9 13.2 ± 0.8 8.8 ± 0.5 21.7 ± 0.913.3 ± 0.6 0.75 ± 0.03 compound² (10% ointment) 4 Solcoseryl 55.11 ±0.76 41.5 ± 1.8 58.5 ± 1.8 15.6 ± 0.4 8.8 ± 0.8 20.0 ± 1.5 14.0 ± 0.70.72 ± 0.06 5 Spasatel 60.62 ± 0.87 43.4 ± 1.6 56.6 ± 1.6 16.0 ± 0.7 9.8± 0.6 18.4 ± 0.8 12.3 ± 0.8 0.77 ± 0.05¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (Compound 2)

TABLE 17 General protein and protein fractions of rats' blood serumAnimal Total Globulins Group Protein Albumin % Globulin % á₁ á₂ â ã A/G15 days 1 Control 58.93 ± 1.29 46.9 ± 1.7 53.1 ± 1.7 10.3 ± 1.0 10.2 ±1.6  18.7 ± 0.4 13.9 ± 0.4 0.89 ± 0.06 (untreated) 2 Placebo¹ 59.22 ±1.65 48.2 ± 2.4 51.8 ± 2.4 10.1 ± 0.3 10.0 ± 1.1  17.3 ± 1.1 14.4 ± 0.60.94 ± 0.08 3 Test 59.06 ± 1.97 48.3 ± 1.7 51.7 ± 1.7 10.3 ± 0.9 7.9 ±0.7 19.6 ± 1.5 13.8 ± 1.1 0.94 ± 0.06 compound² (10% ointment) 4Solcoseryl 57.93 ± 0.51 47.8 ± 0.6 52.2 ± 0.6 11.7 ± 0.9 7.6 ± 0.5 18.9± 0.9 13.9 ± 0.5 0.92 ± 0.02 5 Spasatel 63.24 ± 1.19 47.0 ± 2.4 53.0 ±2.4 11.5 ± 0.9 8.1 ± 0.6 21.5 ± 1.3 11.9 ± 0.8 0.90 ± 0.08¹lanolin ointment base²1-ethyl-3-methyl-4,5-bis (N-methylcarbamoyl)imidazoliumbenzenesulfonate (Compound 2)

When medications are applied to the skin, physicochemical propertiessuch as lipophilicity affect the rate at which the medication isliberated from the composition and absorbed into the skin (Scheuplein R.J, 1965). Our results demonstrate that, as a result of their lowlipophilicity, the salts of N-substituted1,3-dialkyl-4,5-biscarbamoylimidazole derivatives penetrate throughbiological membranes poorly, thus helping to prolong contact with thedamaged area and increasing their efficiency during local application.Therefore these agents are suitable for local application, for exampleas an ointment, cream or gel.

EXAMPLE 15 Morphological assessment of the wound healing stages

An important indicator of wound healing is the morphology andepithelialization of the wound surface (Sarkisov et al, 1981). Sampleswere taken from experimental animals on the fifth, tenth and fifteenthdays of the study described in Example 13, which roughly correspond tothe three wound healing stages: the first traumatic inflammation stageis complete by the fifth day, the granulation tissue development stageis complete by the tenth day, and cicatrisation and epithelializationtake place from the fifteenth to twentieth days. The results areillustrated in FIG. 1.

Day 5

a) Untreated control. The wounds were of significant size, covered bythick pellicles or scabs consisting of necrotic mass, fibrin, andleucocytes, with colonies of bacteria. Only 20% of animals cleaned thewound from the pellicle at individual spots; in the remaining cases thepellicle was closely connected to the wound surface, which was diffuselyinfiltrated by leucocytes. The granulation tissue on the bottom of thewound featured maturing and irregularly located vessels and fibres.There was productive inflammation and oedema in the areas adjacent tothe wound. At the initial stages insignificant epithelialization isfound at the edges of the wound. The degenerate wedge-shapedepithelialization layer has no distinct differentiation features.

b) Placebo. The wound was considerable in size, closely connected to thesurface under the pellicle. There was intensive leucocyte infiltrationof surface areas of the wound bottom, with the pellicle melting at theboundary. The border between the granulation tissue of the wound bottomand adjacent skin and hypoderma was indistinct, with weakly maturingfeatures of the granulation tissue. 60% of the animals had insignificantepithelial regeneration at the edges of the wound, without distinctdifferentiation.

c) Compound 2

The wound was considerable in size, with focal removal of the pellicle,whose thickness was variable but often negligible. The surface was freeof pellicle at the periphery of the wound, where regeneration of theepithelium was observed in all the animals. There was a wedge-shapedlayer of regenerating epithelium of various sizes, with distinct signsof differentiation of flat cells. At the bottom of the wound,granulation tissue matured with inflammatory infiltration, which wasmore pronounced on surface sections. The boundaries between the bottomand the side sections of the wound were indistinguishable, due to oedemaand inflammation.

Day 10

a) Untreated Control group. All of the wounds were covered with a thickpellicle consisting of necrotic mass, fibrin, leucocytes with coloniesof bacteria. There were no signs of cleaning, and the pellicle wastightly bound to the bottom of the wound. On the bottom, there wasmaturing granulation tissue with a large number of newly formed vessels,irregular, chaotically-arranged collagen fibres, and pronouncedleucocyte infiltration. There was diffuse inflammatory infiltration ofthe connective tissue, which is more intensive on the surface areas. In60% of the cases, there were areas of purulent fusion of the connectivetissue. Productive diffusive inflammation resulting in sclerosis wasobserved in the cellular tissue adjacent to the bottom and in theconnective tissue. The inflammation was not limited to the bottom of thewound, and spread to the adjacent areas of the dermis. There was weakinitial epithelial regeneration, with limited wedge-shaped regeneratingareas at the edges of the wound and no distinct signs ofdifferentiation.

b) Placebo. The cleaning of the wound surface was negligibble, andleucocytes were prevalent on the pellicle. In the areas without signs ofcleaning, there was intensive leucocyte infiltration at the bottom ofthe maturing connective tissue. There were areas with a tendency towardscicatrisation of the connective tissue. The wound bottom did not haveclear boundaries: the inflammation was spread over the adjacent dermisand fatty cellular tissue. Epithelial regeneration was virtually absent,and could be observed-only at one or two edges in 60% of the animals.

c) Compound 2

The surface of the wound showed irregular remains of the pellicle,represented by fibrin, erythrocytes and lysing leucocytes. Connectivetissue with correct orientation of collagen fibres and moderate cellinfiltration predominated at the bottom of the wound. The boundaries ofthe regenerating connective tissue were more distinct, and inflammatoryinfiltration of the dermis adjacent to the wound was insignificant.Epithelial regeneration was found in all of the wounds. The regeneratingepithelium was rather extended, with clear signs of multi-layerdifferentiation, and epithelium grew in some areas without completecleaning of the bottom with epithelium growing under the pellicle.

Day 15

a) Untreated Control group. Only 20% of the animals demonstratedsignificant cleaning of wounds and distinct but uncompletedepithelialization: the central part of the wound was devoid ofepithelium, and the bottom had mature connective tissue with a tendencytowards cicatrisation. 80% of the animals had wounds with incompletecleaning of the surface and distinct inflammatory infiltration of themature connective tissue, with uneven cicatrisation on various levels ofthe regenerating connective tissue. Granulation tissue was observed inthe areas of distinct inflammatory infiltration, with leucocytestopographically corresponding to the zones which did not show signs ofcomplete cleaning. Epithelial regeneration was weak, with wedge-shapedextension of the epithelial layer out of the wound edges.Pseudoepitheliomatous hyperplasia was seen in the epidermis of the skinareas adjacent to the wound. This is illustrated in FIG. 1A.

b) Placebo. 60% of the animals showed complete cleaning of the woundsurface and wound epithelialization. However, multi-layer flatregenerating tissue was infiltrated with leucocytes, with oedema andclear signs of dystrophy. These areas correspond topographically to thefoci of pronounced oedema, hyperaemia, inflammatory infiltration of themature connective tissue at the bottom of the wound. Sclerosis andgranulomas of foreign substances were found in the fatty tissue. Theepidermis adjacent to the wound showed pseudoepitheliomatoushyperplasia. 40% of the animals demonstrated weak cleaning, with onlythe edges of the wound being epithelialized, which corresponds withintense inflammatory infiltration of the connective tissue regenerant.

c) Compound 2

60% of the animals showed complete epithelialization of the wound, witha considerable extent of differentiation of the regenerating epitheliumforming a multiple flat layer over the entire wound area. Under themulti-layered flat epithelium, there was mature connective tissue withnormal collagen fibre architecture, and no signs of disorganisation andcicatrisation. Cellular infiltration of the regenerating connectivetissue was minimal. Clear boundaries could be observed between thebottom of the healed wound, sub-epidermal cellular tissue and adjacentareas of dermis. 40% of the animals showed incomplete epithelializationon a considerable area of the bottom of the wound. In this case, therewere patchy remains of pellicle on the surface of non-epithelializedareas. The epithelial regenerant sometimes grew under the pellicle anddetached it from the wound. In such areas, there was focal lymphocyte,leucocyte and macrophage infiltration. This is illustrated in FIG. 1B.

EXAMPLE 16 Morphological indicators of wound healing

A comparative assessment of the healing of a full-thickness wound wasperformed on the basis of a set of morphological indicatorscharacterising the regeneration process:

(a) timeframe and features of wound cleaning, and secondary infection ofthe aseptic wound;

(b) timeframe, extent, direction and the level of differentiation ofepithelial regeneration at the bottom of the wound;

(c) time of development, topography, and spreading of connective tissueregenerant;

(d) presence and degree of inflammation in the regenerant;

(e) presence or absence of synchronisation of the regeneration processesin the epithelium-connective tissue system;

(f) presence or absence of morphological signs of pathologicalregeneration of epithelium in connective tissue (acanthosis,pseudoepitheliomatous hyperplasia, horny cyst, cicatrisation); and

(g) correlation between these processes and the type of wound healing(primary or secondary intention).

Skin wounds were created and treated in all the animals in each group,as described in Example 14. All the indicators listed above variedwithin the group. However, these differences highlighted certaintendencies inherent in the five groups of wounds. Thus the untreatedcontrol group showed the following:

1. Delayed or incomplete cleaning of the massive pellicle (necroticmasses, fibrin, leucocytes) from the bottom of the wound, which wasobserved with some animals even at the fifteenth day of the experiment;

2. Epithelial regeneration was weak and stabilised in the boundary areasof the wound, and epithelial regeneration did not demonstrate a tendencyto differentiation of flat cells;

3. Considerable growth of the granulation tissues on the bottom of thewound, with uneven maturation and consistent inflammatory infiltration;

4. Lack of simultaneous proliferation in epithelium and connectivetissue, which, in conjunction with current inflammation, determines thetendency to healing by secondary intention.

Morphological data obtained in the placebo groups (lanolin ointmentbase) were similar to those in the untreated groups on the fifth andtenth days. However, a positive dynamic was observed on the fifteenthday: the wounds recleaned and epithelialized in a considerable area, andregenerating connective tissue had matured. However, the tendencytowards inflammation remained, and the inflammation spread to theepithelium, which reduced the possibility of epithelial proliferationand differentiation and encouraged wound healing by secondary intention.This is illustrated in FIG. 1A.

Considerable differences were observed when studying the healing ofwounds with the test compound. These differences supplement and confirmthe results on the positive healing effect of Compound 2 onfull-thickness skin wounds. As early as the fifth day, the compoundinduced focal cleaning of the wound surface from the pellicle and theformation of epithelium regenerant with signs of differentiation of flatcells at the wound edges. On the tenth day, simultaneous regeneration ofepithelium and connective tissue and a tendency towards even maturationof regenerant was clearly observed. Even when the pellicle partiallyremained on the wound surface, epithelial regenerant grew under it. Bythe fifteenth day, most animals demonstrated the completion of thehealing process by primary intention, with differentiation of flatcells, and no signs of pathological regeneration or cicatrisation ofconnective tissue. This is illustrated in FIG. 1B.

Spasatel resulted in less favourable healing than Solcoseryl, and wasmarkedly less effective than compound 2. The cleaning of the surface wasslow and incomplete, even on the fifteenth day, and correlated withconstant, extensive inflammatory infiltration of the connective tissueregenerant. In a number of cases, during the inflammation, thesimultaneity of regeneration in the epithelium-connective tissue systemwas disrupted, the surface was incompletely epithelialized, andconnective tissue was cicatrised in some foci, creating prerequisitesfor healing by secondary intention. This is illustrated in FIG. 1C.

Solcoseryl was comparable to Compound 2 in proliferation potential.However, with Solcoseryl the pellicle was detached and the wound wascleaned at a later stage, which created conditions which promotedinflammation in the regenerating connective tissue. This correlated withthe incomplete epithelialization of the wound, and the possibility ofleucocyte degradation of the regenerating epithelium, which createsprerequisites for healing by secondary intention. This is illustrated inFIG. 1D.

EXAMPLE 17 Treatment of wounds in human patients

The effect of Compound 2 on the healing of different types of wounds wastested in human patients. A powder, solution or ointment of thesubstance containing the active agent in a proportion of 5% to 95% wasapplied to the wound. In all cases the wounds healed rapidly withoutinfection or scarring.

1. Patient A. 38 Years Old (Incisional Wound 5 cm)

The wound surface was treated with 10% Compound 2 in a lanolin ointmentbase 12 hours after the wound was made. 1 g of the ointment was appliedunder a bandage. The procedure was repeated daily for 5 days. The woundclosed over, and at 17 days there was no scar, and the skin had fullyrecovered.

2. Patient B. 78 Years Old (Bed Sores on the Thigh 1.0×2.0 cm)

0.5 g of a finely dispersed powder of Compound 2 was applied to thesurface of the wound under a bandage. The procedure was repeated dailyfor 5 days. The wound closed over, and at 16 days there was no scar, andthe skin had fully recovered.

3. Patent C. 15 Years Old (Second Degree Thermal Burn on the Left Hand,2.0×1.5 cm)

A sterile napkin wetted with a 10% sterile aqueous solution of Compound2 was applied to the wound 10 minutes after the burn. The napkin wasrewetted as it dried. The procedure was repeated within 24 hours. In onehour, the pain at the place of burn had stopped. On the next day it wasobserved that hyperaemia had been considerably reduced at the site ofthe burn. Examination at 16 days demonstrated complete recovery of theskin, without scars.

4. Patient D. 9 Years Old (Abrasion of the Left Leg, 3.0×0.5 cm))

0.5 g of a finely dispersed powder of Compound 2 mixed with an equalamount of talc was applied to the surface of the wound under a bandage.The procedure was repeated after 5 days. The wound closed over, and at12 days the skin had healed completely.

5. Patient E. 68 Years Old (Erosion of the Uterine Cervix, 1.5×1.2 cm)

The surface of the wound was dried, and 10% Compound 2 ointment,prepared as for patient A, was applied to the wound surface on a tampon.The procedure was repeated daily for two weeks. A colposcopy at 18 daysshowed that the epithelium of the mucous membrane of the uterine cervixhad fully recovered.

EXAMPLE 18 Effect of Compound 2 on the healing of mouth ulcers

A model of mouth ulcers using ulcers caused by cryogenic injury of theinner surface of the cheek was used for this purpose. Tests wereperformed on 110 male rats weighing 180-200 g, with 10 animals in eachgroup. Ulcers were created in experimental animals by pressing a metalrod of 2.5 mm in diameter cooled in liquid nitrogen to the mucosa for 10seconds under ether anaesthesia. Animals were kept in separate cages,with normal food and water ad libitum. Treatment was administered byspraying of the mucous membrane of the mouth, 24 hours after woundingand then daily until healing was complete. Compound 2 was administeredto the animals at a dose of 50 mg/kg (2 ml of aqueous solution). Thefollowing groups of animals were used:

Group 1: intact animals;

Group 2: cryogenic injury (wound) on day 1;

Group 3: wound on the 3rd day;

Group 4: wound+treatment on day 3;

Group 5: wound on day 7;

Group 6: wound+treatment on day 7;

Group 7: wound on day 14;

Group 8: wound+treatment on day 14.

Cryogenic damage to the oral mucosa results in a wound characterized bya necrotic surface, inflammation with intensive leukocyte infiltration,pronounced blood circulation disorders, fibrinoid necrosis of theconnective tissue of the mucosa and expansion of the inflammation intothe muscle, and reactive skin inflammation.

The progress of wound healing was examined morphologically on days 1, 3,7 and 14. On the day 3 the lesions were progressively worse, resultingin the growth of necrotic changes with the formation of a wide necroticzone at the bottom of the wound, and growth and expansion of acuteinflammation involving the muscle, skin and mucosa beyond the woundarea. On day 7, the wounds in half of the animals were partially clearedof the necrotic mass, accompanied by the development of granulationtissue at the bottom of the wound and in the adjacent mucosa. There wereinitial signs of regeneration of multilayer flat epithelial cells at theedges of the wound, and decreased signs of inflammation. On days 7 and14 in particular, there was an increased tendency towards wound surfacecleaning and connective tissue development and maturation. However,focal inflammatory activity with focal purulent fusion of the maturinggranulation tissue remained in the surface areas of the wound,particularly under the necrotic mass. Epithelialization of the wound didnot exceed the boundaries of the initial stage, taking place at theedges of the wound. There was no sign of a tendency towards completeepithelialization. These results are illustrated in FIG. 2A.

A comparison of the experimental group treated with Compound 2 and thecontrol group demonstrated that on day 3 the wound was free of necroticmass and that there was an increase in granulation tissue, correlatingwith subsiding inflammation, as well as epithelial regeneration at theedges of the wound. Wound healing processes in the experimental groupwere even more distinct on day 7.60% of the animals showed completecleaning of the wound surface, which was accompanied by reducedinflammation, maturation of granulation tissue, reduction of size of thewound defect, as well as more pronounced signs of epithelialregeneration. These results are illustrated in FIG. 2B.

On day 14, these tendencies were more pronounced: there were no woundswith incomplete cleaning of necrosis from the bottom, and there were nosigns of active inflammation on the bottom and adjacent tissue. The sizeof the wound surface was significantly reduced; maturation of thegranulation tissue on the bottom and in the adjacent mucosa correlatedwith a tendency towards complete epithelialization of the wound surfacein all animals, without any scars or deformity. These results areillustrated in FIG. 2C.

Therefore comparative morphological analysis indicated that Compound 2induced a more pronounced, earlier and more complete healing of acryogenic injury of the rat oral mucosa compared to untreated controls.

EXAMPLE 19 Effect of Compounds 1-3 on repair processes in the ratgastric mucosa

Experiments in rats using various models of stomach damage indicatedthat compounds 1, 2 and 3 have a prophylactic and therapeutic effect, assummarized in Table 18. The models used gastric ulcers induced by

a) Electrostimulation of immobilised rats for 3 hours;

b) Immobilization and hypothermia (induced by water cooling);

c) Ulcer created by local wounding with a cryogenic probe using a methodsimilar to that described above for mouth ulcers.

The compounds were administered intraperitoneally at a dose of 20 mg/kg;in the cryogenic ulcer model compound 2 was administered eitherintraperitoneally (10 mg/kg) or orally (20 mg/kg twice daily, ie. atotal of 40 mg/kg/day) and was compared to orally-administeredgastrocepin (1 mg/kg twice daily, ie. a total of 2 mg/kg/day).

However, in contrast to Compound 1, Compound 2 significantly reduced thefrequency, size and severity of the injury of the mucosa. Compound 3 didnot affect gastric secretory function in dogs or rats in either chronicor acute tests.

All three compounds accelerated repair processes in the gastric mucosa.As shown in Table 18, after 3 days rats treated with these compounds had1.2-2 fold less erosive injury than untreated animals.

The cryogenic ulcer model demonstrates that by day 14 Compound 2 at adose of 10 mg/kg reduced the size of lesions in the gastric mucosa by53% when administered intraperitoneally, and by 51% when a daily dose of40 mg/kg was administered orally. The effect of Compound 2 was similarto that of the conventional ant-ulcer agent Gastrozepin (pirenzepine;Thomae).

Both prophylactic and therapeutic administration of Compound 2 toanimals reversed the changes in content of the enzymes cytochromeoxidase and succinate dehydrogenase, whose level is an index of thestatus of the oxidative phosphorylation and functional activity of thegastric mucosa. TABLE 18 PROTECTIVE AND HEALING EFFECT OF COMPOUND 1, 2AND 3 ON LESIONS OF THE GASTRIC MUCOSA IN RATS % % Average reductionreduction number of of of lesions per degree of Area of ulcerated Typesof Lesion animal ulceration lesion area Protective effective 30 minutesbefore the test Electric stimulation of immobilised animals Control 7.0± 0.7 0 15.2 ± 5.7 0 Compound 1 7.0 ± 0.7 0 15.0 ± 4.7 0 Compound 2 2.4± 0.8 66  2.8 ± 2.1 91 Compound 3 3.5 ± 0.4 50  7.1 ± 4.1 54Immobilisation of animals under hypothermia Control 11.5 ± 1.5  0 10.4 ±2.4 0 Compound 1 10.2 ± 1.2  0 10.1 ± 1.4 0 Compound 2 4.1 ± 0.9 64  4.1± 0.8 62 Compound 3 6.8 ± 1.4 41  7.2 ± 2.0 41 Healing effect Electricstimulation of immobilised animals (healing effect over the course of 3days) Control 3.4 ± 0.2 0 Compound 1 2.8 ± 0.7 18 Compound 2 1.7 ± 0.250 Compound 3 2.5 ± 0.4 26.5 Cryogenic ulcer (healing effect over thecourse of 14 days) Control 18.6 ± 2.2  Compound 1 8.8 ± 1.9 53 Compound2 9.4 ± 1.0 62 (twice daily, oral) Compound 3 7.7 ± 2.5 59 (twice daily,oral)

EXAMPLE 20 Clinical studies of the effect of Compound 2 on gastric andduodenal ulcer.

A clinical trial of the effect of Compound 2 on gastric and duodenalulcer was carried out in several gastroenterological clinics in Russia.A total of 167 patients, 30 to 75 years old, was studied; of these 93had duodenal ulcers and 74 had gastric ulcers. The results weremonitored by gastroscopy at weekly intervals. Each patient continued toreceive the treatment which s/he had been given prior to commencement ofthe trial. Compound 2 was administered in addition to the previoustreatment. Compound 2 was given orally as three 0.2 g tablets per dayover a period of three weeks, ie a total dose of 12 g for the treatmentcourse, Compound 2 healed 80% of all the duodenal ulcers treated, and54% of all the gastric ulcers treated. Compound 2 was also effective incases which had been found to be resistant to conventional therapy. Theeffect of Compound 2 was compared with those of a placebo, orcimetidine. Even better results were obtained when Compound 2 wasadministered together with conventional therapy. No side effects wereobserved. Following termination of treatment with Compound 2 noulceration was observed, ie there was no sign of the abstinence syndromewhich is typically seen with H₂-blockers and hormones. Compound 2 was aneffective therapeutic agent with a pronounced reparative effect onerosive and ulcerating injuries of the gastroduodenal area.

EXAMPLE 21 Effect of Compound 2 and Compound 6 in a model of ulcerativecolitis

A model of cryogenic damage of the colon was used in this experiment tosimulate ulcerative colitis. Cryogenic injuries of the sigmoid colon ofrats were induced by pressing a metal rod of 2.5 cm in diameter cooledin liquid nitrogen to the serous membrane of the colon for 10 secondsunder ether anaesthesia.

Tests were performed on 67 male rats weighing 180-200 g, with 8 animalsin each group. Animals were kept in separate cages, with normal food andwater ad libitum. The test compounds were administered orally on thesecond day after the operation, and then daily until healing wascomplete. Compound 2 or Compound 6 was administered to test animals at adose of 20-50 mg/kg. The following agents were used for comparison:

50 mg/kg of Potassium orotate+50 mg/kg of Riboxin (inosine);

180 mg/kg of Methyl uracil;

2 g/kg of Sulfasalazine (Pharmacia)

The following groups of animals were used:

Group 1: intact animals days 1, 3 and 7;

Group 2: ulcer untreated control days 1, 3 and 7;

Group 3: ulcer+Compound 220 mg/kg orally days 3 and 7;

Group 4: ulcer+Compound 250 mg/kg days 3 and 7;

Group 5: ulcer+Compound 620 mg/kg days 3 and 7;

Group 6: ulcer+Potassium orotate 50 mg/kg+Riboxin 50 mg/kg orally days 3and 7;

Group 7: ulcer+(Methyl uracil 180 mg/kg orally) days 3 and 7;

Group 8: ulcer+(Sulfasalazine 2 g/kg orally) days 3 and 7

The nature of the healing of the ulcer injury was examinedmorphologically on days 1, 3 and 7. The morphological analysis showedthat in the control group of animals damage of the colon wall wasproduced, with destruction of the mucosal and muscular layers anddevelopment of a necrotic process with a total lesion of the mucosa;formation of a leukocyte necrotic scab; significant lesion of themuscular membrane; and acute serosal, fibrocytic, and leukocyteinflammation in the damaged area, including the peritoneum. Theinflammatory process extends beyond the injured area into the adjacentareas, and beyond the peritoneum into fatty connective tissue.

In the control animals the healing process has the following dynamics:

On day 1, there was comprehensive injury, with large necrotic surfacesand no signs of rejecting the necrotic mass, and a pronounced diffuseinfiltration of leucocytes into the sub-necrotic zone. The wall of thecolon was totally damaged, with an acute oedema and leucocyteinfiltration. Acute inflammation extended into the peritoneum, adjacentcellular tissue and colonic areas. The edges of the necrotic zone wererepresented by the mucosa of the adjacent areas of the colon.

On day 3, there was a tendency to reject the scab, which was expresseddifferently in various animals; however, there was no instance ofcomplete cleaning from necrosis. There were no signs of epithelialregeneration in the majority of the animals. The damaged areas showedpronounced inflammation, together with increasing granulation andinitial signs of maturation.

On day 7, there was a significant cleaning of necrosis from the bottomof the injured area. The submucosa and muscular layers were replaced bygranulation with different extents of maturity in conjunction withinflammatory infiltration, which remained in both the injured andadjacent parts of the colon. The size of the wound was reduced, andthere was incomplete regeneration of the mucosa without differentiation.In some cases there were focal remnants of the scab. These results areillustrated in FIG. 4A.

In accordance with these results, we have identified the followingcriteria for comparative evaluation of the healing processes in woundareas treated with different agents:

1. Scope of necrotic alterations, and scab cleaning dynamics of thewound;

2. Degree and extent to which inflammation is found in the wound area;

3. Dynamics, quality and synchrony of repair processes—regeneration ofthe epithelium and connective tissue and quantitative completeness ofregeneration.

Animals treated with the agents used for comparison (Methyl uracil,Potassium orotate+riboxin, Sulphasalazine) did not significantly differfrom the control groups on day 3; on day 7 they were characterised by aslower cleaning of necrosis from the bottom of the wound, weakepithelial regeneration, lack of synchrony of epithelial and connectivetissue regeneration, and persisting inflammation compared to controls.On day 7 18% of animals demonstrated complete healing.

A comparison between the experimental groups treated with Compound 2 orCompound 6 and controls clearly demonstrated a number of majordifferences. Beginning on day 3, there was an accelerated rejection ofthe scab from the surface of the wound, and reduction of the intensityof inflammation in the wound area and beyond. On day 7, there was anacceleration in the granulation growth and maturation in the area ofdamage. When Compound 2 at a dose of 20 mg/kg was used, there was atendency on day 7 towards complete regeneration of the epithelium andmucosa and reduction of the wound area due to synchronous regeneration,which was complete in 100% of cases, as illustrated in FIG. 4B, whereasa dose of 50 mg/kg resulted in a reduction of the wound area in only 67%of animals, as shown in FIG. 4C.

All the signs listed above were observed in all animals in the testgroups; however, their extent and timing varied between differentgroups. The optimal indicators of healing were observed when Compound 2was used at doses of 20 mg/kg and 50 mg/kg; they were less pronouncedwith Compound 6.

EXAMPLE 22 Effect of Compound 2 in a model of myocardial infarction

The effect of Compound 2 on myocardial tissue in an experimental modelof myocardial infarction was examined. In this model system neurogenicdystrophy of the myocardium was induced in rats or rabbits by electricalstimulation of the reflexogenic zone of the aortic arch by implantedelectrodes for a period of three hours. This treatment inducesfunctional disturbances of the activity of myocardial muscle, which arecharacterised by changes in the amplitude of the QRS waves and theposition of the ST segment as demonstrated by 12-leadelectrocardiography. These changes are accompanied by changes inbiochemical indicators such as creatine phosphate, cAMP, and calcium ionconcentrations, and changes in the activity of redox enzymes such assuccinate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphatedehydrogenase. At a later stage organic lesions of the myocardium suchas necrotic and fibrosclerotic foci can be observed.

The effect of Compound 2 given intraperitoneally or orally on theseindicators was examined in rats and rabbits following electrostimulationas described above. The effects on levels of creatine phosphate in ratmyocardial are shown in Table 19, and the effects on adenosinetriphosphatase (ATPase), succinate dehydrogenase and glucose-6-phosphatedehydrogenase rabbit myocardium are shown in Table 20. The effects oncAMP and Ca⁺⁺ levels in rabbit myocardial tissue are shown in FIG. 5,and effects on creatine kinase in rabbit myocardium and blood and thelevels of creatine phosphate in myocardium are shown in FIG. 6. TABLE 19Level of creatine phosphate (μM/g) in rat myocardium followingintraperitoneal administration of compound 2. Conditions of IntactCompound 2 Experiments animals Control 10 mg/kg 20 mg/kg Before 1.22 ±0.06 0.50 ± 0.07 0.92 ± 0.08 1.04 ± 0.15 stimulation 3 days after 1.07 ±0.05 0.54 ± 0.05 0.95 ± 0.06 1.07 ± 0.04 stimulation Oral Compound 2 10mg/kg Before 1.10 ± 0.04 0.47 ± 0.04 0.80 ± 0.07 stimulation 3 daysafter 1.19 ± 0.08 0.76 ± 0.08 1.09 ± 0.1  stimulation

TABLE 20 Effect of Compound 2 on activity of enzymes in rabbitmyocardium 48 hours after electrostimulation Experimental Intact WithoutCompound 2 Ethimizol marker animals treatment 10 mg/kg 5 mg/kg ATPase0.57 ± 0.13 0.28 ± 0.12 0.47 ± 0.13 0.43 ± 0.11 (μM/mg/min) P < 0.001 —P < 0.01 P > 0.05 Succinate 46.1 ± 5.8  31.8 ± 6.2  44.2 ± 6.7  39.7 ±7.4  dehydrogenase (optical density units) P P < 0.001 — P = 0.01 P >0.05 Glucose-6- 5.6 ± 0.6 7.1 ± 0.8 6.2 ± 0.7 5.8 ± 0.7 phosphatedehydrogenase (optical density units) P < 0.01 — P > 0.05 P = 0.05Note:P is estimated in comparison with the group of rabbits which were nottreated after electrostimulation.

These results show that Compound 2 is effective both orally andintraperitoneally in minimising myocardial damage in this model. Controlanimals, which did not receive Compound 2 before or afterelectrostimulation, showed a reduction in biochemical indicators ofmyocardial damage, and also showed necrosis, proliferation of themyocardial stroma, and sclerotic centres, ie scar formation. The effectof Compound 2 was comparable to that of Riboxine or levodopa.

EXAMPLE 23 Effect of Compound 2 in patients with myocardial infarction

A clinical trial of Compound 2 was carried out on 100 patients aged from25 to 75 years during the subacute period of myocardial infarction. Eachpatient continued to receive the treatment which s/he had been givenprior to commencement of the trial. Compound 2 was administered inaddition to the previous treatment. A control group receivedconventional treatment, ie. nitrate, anticoagulants and/or hypotensiveagents as appropriate. The results of treatment of these two groups werecompared with groups of patients receiving riboxine and placebo.Compound 2 was administered over a period of three weeks from the 7th to28th days following infarction, and was administered orally at a dose ofthree 0.2 g tablets per day.

The effects of treatment were monitored by 12-lead electrocardiography,using the QRS Scoring System. Haemodynamic parameters and thecontractile function of the myocardium were monitored using a Toshibaultrasound camera model SSH-60A, using conventional methods.

The effect of Compound 2 was also studied in 25 patients aged 43 to 66years suffering from cardiac insufficiency, in comparison with riboxineor a placebo.

In the patients suffering from myocardial infarction, recovery of thecontractile function of the myocardium was more rapid in patientsreceiving Compound 2 than in patients receiving placebo or riboxine. Inpatients suffering from cardiac insufficiency, Compound 2 prevented theprogression of the condition, and prevented dilatation of the leftventricle. No side effects were observed in either group.

EXAMPLE 24 Effect of Compound 2 in patients suffering from myocardialinfarction

The effect of Compound 2 therapy in patients suffering from the subacutephase of myocardial infarction (MI) was assessed. The patients weredivided into two groups. There were no substantial differences betweenthe groups in terms of age and basic features of necrotic foci. Eachpatient continued to receive the treatment which s/he had been givenprior to commencement of the trial. Compound 2 was administered inaddition to the previous treatment. The first group included 63 patientswho received conventional therapy, i.e. nitrates, anticoagulants and/orhypotensive agents as appropriate, and the second group comprised 44patients who were treated with Compound 2 at a dose of 600 mg per dayfrom the 7th to the 28th day after diagnosis of myocardial infarction,in addition to receiving conventional treatment. The control groupincluded 14 healthy men aged from 26 to 42. The average age of patientsin the first group was 50.3 years, and in the second group, 49.5 years.

Repeated myocardial infarctions were diagnosed for 20% of the firstgroup and 25% of the second group. The rate of localization of necroticfoci in the anteroseptal and anterolateral regions of the left ventricleof the heart was 0.47 in the first group and 0.38 in the second group.The inferior and inferiolateral localization of the necrotic foci wasrecorded at a rate of 0.53 for patients in the first group and at a rateof 0.62 for patients in the second group.

Clinical evidence from daily examinations of both groups and the resultsof electrocardiagraphic examination in 12 abductions were assessed overthe entire observation period. A number of analyses were made on the 7thand 28th day of progress of MI, including clinical blood count, plasmabilirubin, sugar, creatinine, cholesterol, β3-lipoproteins, potassiumand sodium, and urine analysis; the activity of the prothrombin complexand the time of re-calcification were also determined. Centralhaemodynamics were examined using the method of body integralrheography, whereby the stroke volume and cardiac indices werecalculated. Indices of left ventricular contractility—terminal diastolicvolume, ejection fraction, and the extent of systolic contraction ofanteroposterior cavity dimensions (% ΔS)— were examined using theechography method using an Aloka SSD-119 apparatus (Japan). Central andintracardiac haemodynamics was investigated on the 1st, 7th; 14th, 21stand 28th days of the disease. Physical examination data showed nosubstantial differences between the groups of patients during theobservation period.

Clinical tests of Compound 2 showed that treatment of myocardial infarctpatients over the course of three weeks (from the 7th to the 28th day ofthe disease), with a dose of 600 mg per day, did not produce anyclinical side effects. Compound 2 therapy resulted in an increase inunconjugated and hence total bilirubin in serum, which does not,however, exceed the upper limit of standard values. Combining Compound 2therapy with conventional therapy of MI patients increased the rate andextent of regeneration of left ventricular myocardial contractility. Ourresults indicate that Compound 2 is useful for treating MI patients inthe subacute phase.

EXAMPLE 25 Comparison of clinical efficacy of Compound 2 and Riboxin inthe subacute phase of myocardial infarction

A total of 94 patients were examined; of these:

Compound 2 was given to 32 patients;

Placebo was given to 14 patients;

Riboxin (inosine) was given to 18 patients;

Conventional therapy as for example 24 was given to 30 patients. Eachpatient continued to receive the treatment which s/he had been givenprior to commencement of the trial. Compound 2 was administered inaddition to the previous treatment.

All the patients underwent standard laboratory tests prior tocommencement of Compound 2 therapy, 14 days after the beginning of thecourse and upon completion of the course. The tests included clinicalblood analysis; general urine analysis; evaluation of creatinine,bilirubin, transaminase activity, blood sugar, cholesterol andβ-lipoproteins, malonic dialdehyde, Schiff bases, zinc, iron, copper,plasma potassium and sodium, prothrombin and fibrinogen; and an ECGexamination in dynamics.

All the patients received conventional therapy, i.e. nitrates,anticoagulants and/or hypotensive agents as appropriate.

Combination therapy included Compound 2 (1st group—32 patients) orRiboxin (2nd group—18 patients). The agents were administered at a doseof 200 mg three times per day.

In addition to an improvement of subjective data, Compound 2 produced apositive effect on the myocardial contractile function, since there wasa clear increase in the ejection fraction from 54.4±1.5% (an the absenceof the agent) to 60.0±0.5% (approx. 6%), in contrast to the group ofpatients who were treated with Riboxin (56.8±1.3%).

The patients treated with Compound 2 showed a tendency toward thenormalization of cholesterol levels at 96.5% and a decrease in the levelof prothrombin to 91.3%, whereas the figures for patients who weretreated with Riboxin were 85.5% and 88.7%, respectively.

The most pronounced therapeutic effect was obtained for patients withrepeated myocardial infarct, in whom cicatrisation took place one or twodays earlier (27 days on the average) in 90% of cases, than patients whowere treated with Riboxin (12% of cases).

A study of the electrolyte composition of blood, in particularmicroelements such as zinc and copper, produced fairly interestingresults. As is known, myocardial infarct causes a decrease in the levelof zinc. Patients who took Compound 2 showed a substantial increase inthe zinc level compared to the Riboxin group, in whom zinc remainedbelow normal levels throughout the entire course of therapy.Furthermore, it was found that during the treatment with Compound 2 thelevel of copper increased proportionally to the increase in the contentof superoxide dismutase, an antioxidant plasma enzyme, which reflectsnormalization of homeostasis. The patients who took riboxin showed nosuch relationship.

Intensification of lipid peroxide (LPO) oxidation processes accompaniedby lower activity of antioxidant systems plays a prominent role in thepathogenesis of ischaemic cardiac disease. All the patients demonstratedan initially high level of LPO indicators (malonic dialdehyde and Schiffbases). Before treatment, all the patients had above normal malonicdialdehyde levels, i.e. 1.3 nmole/ml on average, with the normal levelbeing 1.21 nmole/ml. During treatment with Compound 2 there was a cleardecrease in malonic dialdehyde to 1 nmole/ml on the 21st day. A decreasein the malonic dialdehyde level during treatment with riboxin isconsistent with progression of the myocardial infarct. The level ofSchiff bases before the treatment was 75 conventional units, ie abovenormal (60 conventional units). After Compound 2 therapy, the level ofSchiff bases fell sharply to 40 conventional units, compared to 62.6conventional units after completion of Riboxin therapy. Beforetreatment, superoxide dismutase activity was low in both groups, beingon average 18-20 conventional units. After Compound 2 therapy, thesuperoxide dismutase levels exceeded the norm (23 conventional units),reaching 25 conventional units; after Riboxin therapy, however, thisparameter not only did not increase, but actually fell slightly, to 21.5conventional units.

All the patients who were treated with Compound 2 felt well, and did notdevelop any allergic reactions or side effects.

EXAMPLE 26 Effect of Compound 2 on carbon tetrachloride-induced liverdamage

The prophylactic and therapeutic effects of Compound 2 on liver damagewere examined in a carbon tetrachloride intoxication model. Liverdysfunction was induced by the intravenous injection of a liquidparaffin-based 50% carbon tetrachloride solution at a dose of 0.8 ml per100 g of body mass daily for four days. Inbred male white rats weighing200-220 g were treated with Compound 2 at a dose of 20 mg/kg givenorally by intragastric tube on the eighth day after injection of carbontetrachloride, and daily for four days thereafter. Control animalsreceived carbon tetrachloride only. Animals were sacrificed 7 days afterthe last injection of carbon tetrachloride, when the morphologicalchanges are most pronounced, and samples of liver were taken forhistology.

In the control animals necrosis of the liver parenchyma and destructionof the characteristic architecture were observed; in addition, fattydystrophy and diffuse inflammatory infiltration of the portal stromawere seen. Mitotic figures were not observed. In animals treated withCompound 2, the degree of necrosis was less, and the necrotic zones werelocalized rather than confluent; albuminous dystrophy and centralinflammatory infiltration of the portal stroma predominated. Giantmononuclear, binuclear and polynuclear hepatocytes were found in allsections, and mitotic figures were frequent. Thus compound 2 has aprotective effect on the parenchyma of the liver and increases liverregeneration following carbon tetrachloride intoxication, suggestingthat the compounds of the invention may be useful in the treatment ofchronic active hepatitis. These results are summarised in Table 21.TABLE 21 Comparison of the main morphological indicators of control andexperimental animal groups following intoxication with CCI₄ IndicatorsGroups of animals (arbitrary) Control Compound 2 P Necrosis of  8.2 ±0.24 2.7 ± 0.3 <0.001 hepatocytes Elimination of  1.8 ± 0.2 4.9 ± 0.4<0.001 glycogen from damaged zones Average mitotic 0.0012 ± 0.001 0.021± 0.001 <0.001 index

EXAMPLE 27 Reparative effect of Compound 2, Compound 4, Compound 5, andCompound 6 after partial hepatectomy

Partial hepatectomy was performed using the conventional method (Higginsand Anderson, 1931) by removing the left lateral and central lobes ofthe liver from 80 male white rats, weighing 180-200 g.

Compound 2, Compound 4, Compound 5 or Compound 6 was administered to theexperimental groups intraperitoneally in equimolar doses of 0.1 mM/kgand 0.2 mM/kg. These optimal doses were determined in preliminaryexperiments. The control group received intraperitoneal injections ofsaline solution. The compounds were administered on the day after theoperation and then one injection was given daily over the course of 7days, since maximum growth of the liver mass in rats is observed in thefirst 7 days after partial hepatectomy (Solopaev, 1980).

The effectiveness of the compounds was compared to that of a combinationof non-steroid anabolic regeneration stimulators which are used in thetreatment of liver pathology, namely Riboxin and potassium orotate at adose of 0.2 mM/kg each (Rychnev and Frolov, 1984).

For quantitative evaluation of the regeneration process in the liver thecoefficient of regeneration completion as determined by the formulabelow was used: ${K = {{\frac{P_{1} - P_{2}}{P_{3}} \cdot 100}\%}},$where

P₁=Liver mass 7 days after partial hepatectomy

P₂=Remaining liver mass after partial hepatectomy

P₃=Mass of liver removed

The initial liver mass which is necessary in order to calculate the massof the organ remaining after the operation was calculated based on thetotal body mass of the animal, because liver mass in rats is directlyproportional to the body mass.

The regression equation is as follows:Y=0.036x+2.37,

where x=mass of the animal, and y=liver mass The coefficient of linearcorrelation for evaluation of the size being investigated is 0.654(Gaivoronskaya et al, 2000).

Stimulation of the regeneration of tissue after hepatectomy in responseto different medicinal preparations is associated with an increase inthe synthesis of nucleic acids, as indicated by the increase in thecontent of RNA and DNA in the regenerated tissue. Quantitative analysisof DNA and RNA was performed by centrifugation. Comparative analysis ofthe content of DNA in healed animals was found to be the mostinformative. The enzymic component of anti-oxidant protection was alsoassessed on the basis of the activity of catalase andsuperoxidedismutase (SOD).

RNA and DNA Content

5 ml of 0.3 MM/kg of HCIO₄ solution were added to the sample (100 mg).In order to ensure complete sedimentation of the acid-insolublefraction, the beakers were put in ice for 15 minutes. Then they werecentrifuged for 10 minutes at 5000 revolutions per minute. The sedimentwas washed twice with 0.2 M HCIO₄. After the final centrifugation, thewalls of the beaker were carefully dried with gauze and filter paper.The sediment was ground down with a glass pestle, suspended in 1 ml ofwater and then 1 ml of 0.6 M KON solution was added at room temperature.Hydrolysis was performed over the course of 1 hour at 37° C., then thebeakers were put in ice to stop the hydrolysis process. 4 ml of the 0.6M solution were added to each beaker and the beakers were left in icefor another 15 minutes, then centrifuged for 15 minutes at 5000revolutions per minute. The supernatant was used to determine the RNA byUV absorption at 260 nm and 290 nm compared to the control samplecontaining 0.4M of HCIO₄). The amount of RNA in μg per 1 ml of thesupernatant was calculated using the following formula:$C = {\frac{D_{270} - D_{290}}{0.19} \times 10.5}$where C=is the RNA concentration (μg/ml)

DNA analysis was carried out on the sediment remaining after thealkaline hydrolysis used to determine RNA. 0.5 M of HCIO₄ (5 ml persample) was poured into the dried beaker containing the sediment.Hydrolysis was performed in a boiling water bath for 20 minutes. Theamount of DNA in μg per 1 ml of the hydrolysate was calculated using thefollowing formula: $C = {\frac{D_{270} - D_{290}}{0.19} \times 10.1}$where C=is the DNA concentration (μg/ml).Catalase Activity

Catalase activity was determined using the permanganate method. Theoptimal amount of the substrate (3 ml of 1% H₂O₂) and 1 ml of the enzymewere added to 10 ml of phosphate buffer, pH 7.8. After 10 minutes thereaction was stopped by adding 1.5 ml of H₂SO₄ (50%). The remaininghydrogen peroxide was filtered with 0.1M of KMnO₄ solution. Catalaseactivity was calculated on the basis that 1 ml of 0.1M of KMnO₄ solutioncorresponds to 1.7 ml of H₂O₂.

Superoxide Dismutase Activity

SOD activity was determined using the dianisidine method, which is basedon the fact that under ultraviolet irradiation Riboflavin (Rb) isconverted to an excited (ionised) state and therefore becomes able toattack reduced dianisidine (DH₂). The resulting flavin semiquinonefurther reduces the molecular oxygen and creates a superoxide radical.In the absence of SOD, the superoxide radical reduces the dianisidineradical in the reaction mixture. When SOD is available, theconcentration of O₂ is very small; therefore dianisidine radicalsinteract with each other, producing one molecule of reduced (colourless)dianisidine and another molecule of oxidised (coloured) dianisidine. Thehigher the SOD activity, the more oxidised dianisidine is produced, withmaximum absorption at 460 nm.

Incubation Medium: 0.1 M phosphate buffer 1 ml Riboflavin 0.26 mlo-dianisidine 0.04 ml Sample 0.1 ml

The control sample contains a known amount of SOD. The samples wereexposed to UV light for 10 minutes from a distance of 10 cm, cooled atroom temperature and the optical density was measured.

The results, which are summarized in Table 22, showed that hepatectomyresulted in a distinct increase in DNA and RNA in the rat liver. The DNAcontent in the group treated with Compound 2 at a dose of 0.1 mM/kg or0.2 MM/kg was 15% higher than that in animals undergoing hepatectomyalone, and the regenerating activity of the groups treated with Compound3, Compound 5, Compound 6 or Compound 7 was higher than that of thegroup treated with 0.2 MM/kg Potassium orotate+Riboxin. Theeffectiveness of the compounds was in the following order: Compound 20.1 MM/kg>Compound 2 0.2 MM/kg>Compound 6 0.2 mM/kg>Compound 5 0.2MM/kg>Compound 4 0.2 MM/kg>{Potassium orotate+Riboxin} 0.2 MM/kg.

The level of anti-oxidant protection was also assessed in terms ofcatalase and superoxidedismutase (SOD) activity. The importance of theseenzymes is due to their physiological role related to oxidationprocesses involving molecular oxygen, hydrogen peroxide and oxygenradicals in metabolic changes. The changes in anti-oxidant activity aremore pronounced with regard to catalase. A distinct stabilization ofcatalase activity was observed after treatment with Compound 2 at a doseof 0.2 MM/kg and Compound 6 at a dose of 0.2 MM/kg. These results weresimilar to those of the standard treatment group (Potassiumorotate+Riboxin). An increase in repair processes was also observed withother compounds; however, their efficiency was slightly lower (Compound2, 0.1 MM/kg>Compound 2, 0.2 MM/kg>Compound 5, 0.2 MM/kg>Compound 4, 0.2M M/kg).

Therapeutic efficacy may be assessed in terms of the changes in thesecond anti-oxidant protection enzyme, SOD. Its activity in the liver ofthe experimental animals is reduced after hepatectomy. Administration of0.2 MM/kg of Compound 2 or 0.2 MM/kg of Compound 6 resulted in a morepronounced stimulation of repair processes in the liver than when aconventional treatment (0.2 MM/kg Potassium orotate+Riboxin) was used.Positive changes in SOD activity were also found in the groups ofanimals which received Compound 2 at 0.1 MM/kg or 0.2 MM/kg and Compound4 at 0.2 MM/kg after hepatectomy.

Therefore the efficacy of the compounds of the invention in terms oftheir ability to stabilize anti-oxidant enzyme activity is as follows:Compound 6 0.2 MM/kg>Compound 2 0.2 MM/kg>Compound 5 0.2 MM/kg (seeTable 22). TABLE 22 THE EFFECT OF 1,3-DIALKYL-4,5-BIS(N-METHYLCARBAMOYL) IMIDAZOLIUM SALTS (COMPOUNDS III, V, VI AND VII) ONTHE REGENERATION OF LIVER AFTER A PARTIAL HEPATECTOMY Groups of animalsHepatectomy + test components Hepatectomy Potassium Intact aloneCompound 2 Compound 2 Compound 5 Compound 6 Compound 4 orotate + animals(control) 0.1 mM/kg 0.2 mM/kg 0.2 mM/kg 0.2 mM/kg 0.2 mM/kg RiboxinIndications n = 7 n = 7 n = 7 n = 7 n = 7 n = 7 n = 7 0.2 mM/kg Level of— 100 150.5 134.9 120.3 105.0 128.3 144.7 recovery* RNA content 5.42 ±0.17  7.23 ± 0.25  7.07 ± 0.18  6.96 ± 0.39 7.46 ± 0.14 7.41 ± 0.16 7.14± 0.2 6.95 ± 0.35 (mg/g of tissue) DNA content 2.99 ± 0.21  3.65 ± 0.12 4.2 ± 0.37  4.1 ± 0.13 3.76 ± 0.29 3.88 ± 0.17  3.72 ± 0.09 3.48 ± 0.19(in mg/g of tissue) Catalase E 34.0 ± 1.86 14.0 ± 0.9 18.0 ± 3.1  20.0 ±1.92 17.3 ± 2.6  20.0 ± 2.7  18.0 ± 2.6 23.0 ± 3.1  activity μgM/min/gtissue × 1000 SOD 90.8 ± 1.35 72.7 ± 5.9 78.9 ± 5.8 98.0 ± 4.6 103.0 ±4.4  100.0 ± 4.7  90.0 ± 3.3 80.36 ± %.7   activity Units/g tissueProtein 107.0 ± 4.9  93.2 ± 7.0 86.4 ± 9.8 94.8 ± 8.4 86.8 ± 6.9  93.0 ±14.0  85.5 ± 10.6 91.5 ± 8.5  content Mg/g tissue*After partial hepatectomy some hepatomegaly is common.

EXAMPLE 28 Morphological study of the liver after partial hepatectomyand administration of Compound 2, Compound 4, Compound 5 and Compound 6

Morphological methods are highly informative in the assessment of repairprocesses. Histological tests of the liver after partial hepatectomywere performed using generally accepted methods. Microscopic sectionswere stained with hematoxylin and eosin using Van Gieson's methodology.Liver tests after partial hepatectomy were performed on 8 groups ofanimals, totalling 37 rats. All animals were examined one week afterhepatectomy.

Histological examination indicated that the liver completely regeneratedafter partial hepatectomy in response to the test compounds. Thefollowing compounds were tested and showed no undesirable effects:Compound 2 0.1 MM/kg, Compound 6 0.2 MM/kg, Compound 4 0.2 MM/kg and,for comparison, a standard combination of Potassium orotate+Riboxin 0.2MM/kg.

In contrast to the standard combination and in addition to hypertrophyof hepatocytes, test groups demonstrated an expansion of the growthareas with regenerating nuclei emerging in peripheral areas andproliferation of bile-duct epithelium. However, when Compound 2 0.2MM/kg and Compound 5 MM/kg were used, some of the animals demonstratedhepatocyte hypertrophy combined with subacute hepatitis, with apronounced sclerogenic change in the portal tracts and inflammatoryinfiltration correlating with dystrophic changes in hepatocytes.

The results indicate that Compound 2, which increases the organregeneration completeness ratio by 34.9-50.5%, is the most effective ofthe compounds in activating repair processes in the liver, and exceedsthe DNA content indicators in hepatectomised animals by 15%. The groupsof animals treated with Compound 2, Compound 4, Compound 5 and Compound6 had the highest regeneration activity compared to the combination ofPotassium orotate+Riboxin. In terms of their effectiveness, thesesubstances were as follows: Compound 2 0.1 MM/kg>Compound 2 0.2MM/kg>Compound 6 0.2 MM/kg>Compound 5 0.2 MM/kg>Compound 5 0.2MM/kg>{Potassium orotate+Riboxin} 0.2 MM/kg.

Our results in this model indicate that compounds of the invention areuseful in stimulating liver regeneration following surgical resection.

EXAMPLE 29 Study of the clinical effect of Compound 2 in patients withchronic active viral hepatitis

45 patients aged between 18 and 32 years old with chronic active viralhepatitis were divided into 4 groups and kept under observation. Allpatients had received conventional therapy, usually potassium orotate,riboxin and interferon prior to commencement of the trial.

The first group (10 patients) received conventional therapy for 20 days.Assessment of clinical and biochemical indicators and serology data wascarried out repeatedly for 20 days after the beginning of treatment.

The second group (10 patients) received both conventional treatment andprednisolone at a dose of 15 mg/kg for 20 days. In this group theclinical-biochemical and serological analysis took place before thetreatment, and 10 days and 20 days after the beginning of treatment.

The third group (10 patients) received both conventional treatment andCompound 2 administered orally at a dose of 600 mg a day for 20 days.The control clinical-biochemical and serological analysis was carriedout 20 days after the beginning of treatment.

The fourth group (15 patients) received prednisolone either orally orintravenously at a dose of 15 mg a day for 10 days; then prednisolonewas stopped and replaced by Compound 2 at a dose of 600 mg a day for 10days. In this group the first clinical-biochemical and serologicalanalysis was carried out prior to the beginning of treatment, the secondwhen treatment with prednisolone ceased and the final analysis after thefull course of treatment with Compound 2.

Under such treatment with prednisolone a “rebound” phenomenon isobserved, with an increase in immunoreactivity and a deceleration ofreplication of the virus. Prednisolone is conventionally prescribed at adose of 20-30 mg a day over the course of 4 weeks, then it is abruptlystopped and after several days α-interferon is used. However, whenprednisolone is employed over a long enough period of time (more than 2weeks), replication of viruses is significantly activated, which makesthe disease prognosis unfavourable.

Viral markers were found in all of the patients under observation.Chronic active hepatitis was diagnosed on the basis of primary clinicaland laboratory data: the presence of astenovegetative syndrome,hepatomegaly with sclerosis of the liver, palpitations and tenderness,as well as spontaneous pain in the liver area, dyspeptic disturbancesand icteric sclera. The following biochemical indicators were alsoconsidered: increased alanine transferase activity over the previous 6months; increase in bilirubin or γ-globulin content in serum; as well asa decrease in sublimate titre and albumin content in the serum.

Our results with Compound 2 in patients with chronic active viralhepatitis have shown that this agent improves the clinical picture andnormalizes biochemical indicators, particularly when combined with apreliminary course of prednisolone therapy; furthermore, it makesdyspepsia less pronounced and frequent, and consistently reduces alaninetransferase activity.

EXAMPLE 30 Effect of Compound 2 on repair of bone tissue

A round or oval defect of the bone plate with a diameter of 2.5 mm wasproduced in the bone tissue of the rat lower jaw, using a dental drillunder ether anaesthesia. In the post-operative period, the animals werekept in separate cages, with normal food and water ad libitum. Theexperiments were performed on 130 male rats weighing 180-200 g, with 10rats in each group.

The animal test groups were treated with Compound 2 (50 or 100 mg/kg) ormethyl uracil (50 mg/kg) 24 hours after the damage and on a daily basisfor three months. The control group used animals with injured bonetissue, which did not receive any treatment. The following groups wereused:

Group 1 intact animals;

Group 2 controls (bone plate defect; untreated) 30 days;

Group 3 bone plate defect+Compound 250 mg/kg 30 days;

Group 4 bone plate defect+Compound 2100 mg/kg 30 days;

Group 5 bone plate defect+Methyl uracil 50 mg/kg 30 days;

Group 6 controls (bone plate defect; untreated) 60 days;

Group 7 bone plate defect+Compound 250 mg/kg 60 days;

Group 8 bone plate defect+Compound 2100 mg/kg 60 days;

Group 9 bone plate defect+Methyl uracil 50 mg/kg 60 days;

Group 10 controls (bone plate defect; untreated) 90 days;

Group 11 bone plate defect+Compound 250 mg/kg 90 days;

Group 12 bone plate defect+Compound 2100 mg/kg 90 days;

Group 13 bone plate defect+Methyl uracil 50 mg/kg 90 days;

The repair pattern of the injured bone was assessed morphologically ontissue sections after 30, 60 and 90 days. The sections were examined bylight microscopy at magnifications of ×15, ×100, ×200, and ×400.

Morphological analysis showed that bone tissue in the defect zonesregenerated in response to Compound 2 or methyl uracil by way of primaryhealing with osteogenesis, following the pattern of appositional growthof mature compact bone tissue. There was not a single case of woundinfection resulting in changes in the nature and timing of bone defecthealing. The timing and nature of bone defect healing are consistentwith literature data on bone tissue regeneration phases.

The best results in closing bone defects were shown by the group ofanimals who were given Compound 2 at 50 mg/kg over the course of threemonths, in which the defect was almost fully closed by the newly formedprimary callus bone tissue, as shown in FIG. 7B, as compared withcontrols (FIG. 7A) and other test groups (FIGS. 7C and 7D).

EXAMPLE 31 Toxicity of 1,3-dialkyl-4,5-bis(N-methylcarbamoyl)imidazoliumsalts

The acute toxicity of 1,3-dialkyl-4,5-bis (N-methylcarbamoyl)imidazoliumsalts was assessed following intraperitoneal injection into 200 whitemale mice weighing 18 to 20 g. Dead animals were counted daily after theadministration of the test substance. The total observation period was14 days. LD₅₀ was calculated in accordance with Kerber's method(Belenky, 1963), and the results are shown in Table 23. Death was causedby respiratory failure; when a toxic dose of medication is administered,the animals lie motionless, then stop breathing and suffer cardiacarrest.

Compounds 1, 3, 4 and 7 all had very low toxicity, while even Compound 3had an LD50 of over 500 mg/kg. TABLE 23 Acute toxicity of1,3-dialkyl-4,5-bis(N-methylcarbamoyl)- imidazolium salts (LD₅₀) LD₅₀Medication mg/kg Compound (1) 1833 Compound (2) 764 Compound (3) 1292Compound (4) 1221 Compound (7) 1221

We found that compound 2 is effective at doses of 10 to 20 mg/kg forprolonged oral and/or parenteral administration. In these studies, theLD₅₀ for rats was 1210 mg/kg, and for mice, the LD₅₀ was 1920 mg/kg whengiven intraperitoneally. For oral administration the LD₅₀ for rats andguinea pigs was more than 20000 mg/kg. It is contemplated that inclinical use, treatment using oral tablets will take two to three weeks,with each patient receiving three tablets containing 0.6 g activecompound per day.

Chronic toxicity studies in animals over a period of six monthsindicated that compound 2 had no irritating or allergic action, does notact on the central nervous system, and has no teratogenic, mutagenic orcarcinogenic immunosuppressive activity. No effect on body weight,weight of internal organs, structure or function of internal organs orblood indicators was observed. These studies were performed in mice (40mg/kg per day, given subcutaneously), in rats (40 mg/kg per day, givensubcutaneously) and in dogs (10 mg/kg or 40 mg/kg per day, givenorally).

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

REFERENCES

-   Belenky M. L. Elements of quantitative assessment of pharmaceutical    effect (1963)-   Gaivoronskaya V. V., Okovity S. V., Shustov E. B., Smirnov A. B.,    Experimental and clinical Pharmacology, 2000, T 63, No. 5, P. 34-36-   Higgins G. M. and Anderson R. M., Arch. Pathol, 1931, V.12. N.1, P.    188-202-   Katzung G. B., Dermatological pharmacology /Basic and clinical    pharmacology: 1998 Volume 2 pp. 552-571-   Kuzin M. I., Kostyuchenko B. M., Karlov V. A. Summary review of    wound related theories. //Wounds and wound infection.—M., Medicine,    1981, pp. 13-54-   Liubimov, B. I. et al 1999. Methodological instructions with regard    to the assessemnt of the allergic characteristics of pharmacuetical    substances in “Manual on Experimental Pre-chemical study of new    pharmaceutical substances p 234-241-   Mashkovsky M. D., Medicinals stimulating metabolic processes//    Medicinals. 1998, vol. 2, pp. 168-192-   Piotrovsky L. B., Marasanova N. Y., Khromov-Borisov N. V., Moreva E.    V., Sapronov N. S., Novikov V. P., Kovaleva V. A. Russian Patent No.    1075668: Benzenesulphonates of 1,3-dialkyl-4,5-bis    (N-methylcarbamoyl)-imidazolium, stimulating tissue energy exchange-   Rychnev V. E., Frolov V. M., Regeneration stimulators used in    treatment of viral hepatitis and other liver diseases, Voronezh    State University Press, Voronezh, 1984-   Sarkisov D. S., Paltsyn A. A., Musikant L. I.: Morphology of the    wound healing process, Wounds and wound infection. Moscow.: 1981,    pp. 688-695-   Scheuplein R. J.: Mechanism of percutaneous absorption. J. Investig.    Dermatology, 1965, v.45, N5, P. 334-345-   Solopaev B. P., Regeneration of normal and pathological alterations    of the liver, Volgo-Viat. Book Publishers, Gorky 1980-   Swingle, K. F., Shideman, F. E., 1972 J. Pharmacol. Exp. Therap. 183    226-234-   Sriprachya-Anunt S., Fitzpatrick R. E., Goldman M. P., Smith S. R.    Infections complicating pulsed carbon dioxide laser resurfacing for    photoaged facial skin.//Dermatologic Surgery, 1997, 23(7): 527-35-   Winter, C. A., Risley F. A. Nuss, G. N., 1962 Proc. Natl. Exp. Biol.    Med. 3 544-547-   Zapadnyuk I. P., Zapadnyuk V. I., Zakharia E. A., Zapadnyuk B.    V.//Laboratory animals. -Kiev. Vyscha shkola, 1983, pp. 248-250,    254-255

1. A method of promoting tissue repair or wound healing, comprising thestep of administering an effective amount of a 1,3-dialkyl-4,5-bis(optionally N-substituted carbamoyl)imidazolium salt to a subject inneed of such treatment.
 2. A method according to claim 1, wherein saidmethod reduces inflammation in said subject.
 3. A method according toclaim 1, in which the 1,3-dialkyl-4,5-bis (optionally N-substitutedcarbamoyl)imidazolium salt is a compound of formula I

in which R¹ and R² are the same or different, and each is selected fromthe group consisting of hydrogen and a linear or branched alkyl group of1 to 6 carbon atoms, which may optionally be substituted by an amino,substituted or unsubstituted aminomethyl, nitro, hydroxyl, halogen,carboxy, or carboxylic acid amide group; R³ and R⁴ are the same ordifferent, and each is a substituted or unsubstituted linear or branchedalkyl group of 1 to 6 carbon atoms; and X⁻ is a pharmaceuticallyacceptable inorganic or organic anion selected from the group consistingof chloride, bromide, iodide, sulphate, nitrate, phosphate, perchlorate,formate, acetate, fumarate, malate, malonate, citrate, benzoate,salicylate, benzenesulphonate, methylsulphonate, p-toluenesulphonate,gentisate, and naphthalene-8-sulphonate.
 4. A method according to claim3, in which at least one of R³ and R⁴ is unsubstituted.
 5. A methodaccording to claim 4, in which both R³ and R⁴ are unsubstituted.
 6. Amethod according to claim 4, in which where R¹ or R² is substituted witha substituted sulphonamide, the substituent is an alkyl chain of 1 to 6carbon atoms.
 7. A method according to claim 3, in which R¹ and R² aredifferent; and R³ and R⁴ are the same or different, and is eachindependently an alkyl group with 1 to 6 carbon atoms.
 8. A methodaccording to claim 7, in which R³ and R⁴ are both alkyl groups of 1 to 4carbon atoms.
 9. A method according to claim 8, in which R³ and R⁴ areboth methyl or both ethyl, or one of R³ and R⁴ is methyl and the otheris ethyl.
 10. A method according to claim 8, in which R³ is methyl andR⁴ is ethyl.
 11. A method according to claim 3, in which X⁻ isbenzenesulfonate, benzoate, salicylate, or gentisate.
 12. A methodaccording to claim 11, in which X⁻ is benzenesulphonate.
 13. A methodaccording to claim 3, in which X⁻ is an inorganic anion selected fromthe group consisting of chloride, bromide, and iodide.
 14. A methodaccording to claim 3, in which the subject is suffering from epithelialdamage to skin or mucous tissue, caused by erosions, ulcers, chronicinjury, infection, trauma or surgery.
 15. A method according to claim 1,in which the subject is suffering from a condition selected from thegroup consisting of traumatic wounds, surgical wounds, burns, dehiscedsurgical incisions, grafts, diabetic ulcers, varicose ulcers, decubitusulcers (bedsores), trophic ulcers, tropical ulcers, steroid ulcers,indolent ulcers, oral or pharyngeal ulcers, aphthous ulcers, and cornealulcers; and cervical erosions.
 16. A method according to claim 1, inwhich the subject is suffering from a condition selected from the groupconsisting of gastric or duodenal ulcers, and ulcerative colitis.
 17. Amethod according to claim 1, in which the subject is suffering from acondition selected from the group consisting of myocardial damage, liverdamage and bone damage.
 18. A method according to claim 17, wherein saidmethod stimulates liver regeneration in said subject.
 19. A methodaccording to claim 15, wherein said method reduces or prevents scarformation in said subject.
 20. A method according to claim 16, whereinsaid method treats ulcerative colitis in said subject.
 21. A methodaccording to claim 15, wherein said method treats oral or pharyngealulceration in said subject.
 22. A method according to claim 17, whereinsaid method treats hepatic cirrhosis or chronic active hepatitis in saidsubject.
 23. A method according to claim 16, wherein said method treatsgastric or duodenal ulcers in said subject.
 24. A method according toclaim 17, wherein said method treats myocardial infarction in saidsubject.
 25. A method according to claim 17, wherein said methodstimulates bone repair in said subject.
 26. A method according to claim1, in which the 1,3-dialkyl-4,5-bis (optionally N-substituted carbamoyl)imidazolium salt is selected from the group consisting of1,3-dimethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzenesulfonate,1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzenesulfonate,1,3-diethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzenesulfonate,1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzoate,1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium salicylate,1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium gentisate, and1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium chloride.
 27. Acompound of formula I

in which R¹ and R² are the same or different, and each is selected fromthe group consisting of hydrogen and a linear or branched alkyl group of1 to 6 carbon atoms, which may optionally be substituted by an amino,substituted or unsubstituted aminomethyl, nitro, hydroxyl, hydrogen,carboxy, or carboxylic acid amide group; R³ and R⁴ are the same ordifferent, and each is a substituted or unsubstituted linear or branchedalkyl group of 1 to 6 carbon atoms; and X⁻ is a pharmaceuticallyacceptable inorganic or organic anion selected from the group consistingof chloride, bromide, iodide, sulphate, nitrate, phosphate, perchlorate,formate, acetate, fumarate, malate, malonate, citrate, benzoate,salicylate, benzenesulphonate, methylsulphonate, p-toluenesulphonate,gentisate, and naphthalene-8-sulphonate, with the proviso that when X⁻is benzenesulphonate, R¹ is hydrogen and R² is methyl, R³ and R⁴ are notmethyl or ethyl.
 28. A compound according to claim 27, in which at leastone of R³ and R⁴ is unsubstituted.
 29. A compound according to claim 28,in which both R³ and R⁴ are unsubstituted.
 30. A compound according toclaim 27, in which where R¹ or R² is substituted with a substitutedsulphonamide, the substituent is an alkyl chain of 1 to 6 carbon atoms.31. A compound according to claim 27, in which R¹ and R² are different;and R³ and R⁴ are the same or different, and are each independently analkyl group with 1 to 6 carbon atoms.
 32. A compound according to claim31, in which R³ and R⁴ are alkyl groups of 1 to 4 carbon atoms.
 33. Acompound according to claim 32, in which R³ and R⁴ are both methyl orboth ethyl, or one of R³ and R⁴ is methyl and the other is ethyl.
 34. Acompound according to claim 33, in which R³ is methyl and R⁴ is ethyl.35. A compound according to claim 27, in which X⁻ is benzenesulfonate,benzoate, salicylate, or gentisate, with the proviso that when X⁻ isbenzenesulphonate, R¹ is hydrogen and R¹ is methyl, R³ and R⁴ are notmethyl or ethyl.
 36. A compound according to claim 35, in which X⁻ isbenzenesulphonate.
 37. A compound according to claim 27, in which X⁻ isan inorganic anion selected from the group consisting of chloride,bromide, and iodide.
 38. A compound according to claim 27, selected fromthe group consisting of1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium benzoate,1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium salicylate,1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium gentisate, and1-methyl-3-ethyl-4,5-bis(N-methylcarbamoyl)imidazolium chloride.
 39. Acompound according to claim 27, together with a pharmaceutically orveterinarily acceptable carrier.
 40. A compound according to claim 39,in which the carrier is adapted for topical administration.
 41. Acompound according to claim 39, in which the carrier is adapted fororal, buccal or sub-lingual administration.
 42. A method of synthesis ofa compound according to claim 27, comprising the step of subjecting a1-alkylimidazole-4,5-bis(optionally N-substituted carbamoyl)imidazole toalkylation (quaternization) with an alkyl benzenesulfonate to producethe corresponding imidazolium benzenesulfonate, and optionally replacingthe benzenesulfonate anion by ion exchange, in which the imidazolemoiety is as defined in claim
 27. 43. A method of promoting tissuerepair, promoting wound healing or reducing inflammation in a subject,comprising administering to said subject a 1,3-dialkyl-4,5-bis(optionally N-substituted carbamoyl)imidazolium salt in an amounteffective to promote tissue repair, promote wound healing or reduceinflammation in said subject.
 44. A composition comprising a compoundaccording to claim 27 and a pharmaceutically or veterinarily acceptablecarrier. 45-50. (canceled)